Plant Tissue Culture and CRISPR: A Synergistic Framework for Precision Plant Biotechnology

Abstract

This article provides a comprehensive overview of the integral role of plant tissue culture in the development and application of CRISPR-based genome editing for plant biotechnology. It covers foundational principles, from the establishment of disease-free explants to the mechanisms of CRISPR/Cas systems, and details advanced methodological workflows for stable transformation and transgene-free plant regeneration. The content addresses key challenges such as editing efficiency, delivery methods, and genotypic dependence, offering targeted optimization strategies. Furthermore, it explores rigorous validation techniques and comparative analyses of editing tools, positioning the synergy of tissue culture and CRISPR as a transformative platform for developing novel plant traits with significant implications for agricultural and biomedical research.

The indispensable role of plant tissue culture in the CRISPR editing workflow

Plant tissue culture (PTC) serves as the foundational platform for regenerating whole plants from genetically engineered cells, making it an indispensable component in modern crop improvement pipelines. For CRISPR-edited plants, the regeneration of a single, transformed cell into a fertile plant is arguably the most critical determinant of success. This application note details a highly efficient, five-stage protoplast regeneration protocol recently developed for the oilseed crop Brassica carinata, achieving up to 64% regeneration frequency and 40% transfection efficiency [1]. We contextualize this protocol within the broader framework of overcoming recalcitrance in CRISPR/Cas9 workflows, providing researchers with a standardized, high-yield methodology that can be adapted for other economically important species.

The CRISPR/Cas9 system has revolutionized plant biotechnology by enabling precise, targeted genome modifications. However, a significant bottleneck persists: after the CRISPR machinery is delivered and edits are made in plant cells, the entire, fertile plant must be regenerated from those few modified cells [2] [3]. This regeneration is almost exclusively dependent on plant tissue culture techniques. Many crucial crops, including staples like wheat and maize and most woody trees, are "recalcitrant," meaning they resist regeneration in vitro, severely hampering genetic improvement efforts [4] [3].

The synergy between tissue culture and CRISPR is powerful yet challenging. While CRISPR acts as a precise genetic scalpel, tissue culture is the essential recovery room that nurtures a single edited cell into a whole organism [3]. This note presents an optimized regeneration protocol designed to function as a robust and efficient "gateway," specifically tailored to integrate seamlessly with CRISPR genome editing projects, thereby accelerating both basic research and commercial crop development.

Featured Protocol: Highly Efficient Protoplast Regeneration forBrassica carinata

This protocol is adapted from a 2025 study that systematically investigated factors influencing in vitro shoot regeneration, including genotype, sugar type, and the critical selection and combination of plant growth regulators (PGRs) [1].

Plant Material and Protoplast Isolation

• Plant Material: Seeds of Brassica carinata (e.g., advanced line S-67 x Holetta-1, cultivar 'Derash') are surface-sterilized and germinated on half-strength Murashige and Skoog (MS) medium [1].
• Protoplast Isolation:
  • Harvest fully expanded leaves from 3- to 4-week-old seedlings.
  • Slice leaves finely and incubate in plasmolysis solution (0.4 M mannitol) for 30 minutes in the dark.
  • Digest tissue in an enzyme solution containing 1.5% cellulase Onozuka R10, 0.6% Macerozyme R10, and 0.4 M mannitol for 14-16 hours in the dark with gentle shaking.
  • Purify the protoplast suspension by filtration through a 40-µm nylon mesh and repeated centrifugation (100 g) in W5 solution [1].

The Five-Stage Regeneration Protocol: Media and Hormonal Control

The core of this protocol is a five-stage system, each with a distinct hormonal objective. The specific PGR concentrations are summarized in Table 1.

Table 1: Plant Growth Regulator (PGR) Requirements for the Five-Stage Protoplast Regeneration Protocol of Brassica carinata [1].

Stage	Medium	Key Objective	Auxin Requirement	Cytokinin Requirement	Critical PGRs & Notes
1	MI	Cell Wall Formation	High	Not Required	High concentrations of NAA and 2,4-D.
2	MII	Active Cell Division	Lower than MI	Required	Lower auxin (NAA) relative to cytokinin (BAP).
3	MIII	Callus Growth & Shoot Induction	Low	High	High cytokinin-to-auxin ratio.
4	MIV	Shoot Regeneration	Very Low	Very High	Even higher cytokinin-to-auxin ratio than MIII.
5	MV	Shoot Elongation	Not Required	Very Low	Low levels of BAP and GA3 for shoot development.

The logical sequence and hormonal shifts of this protocol are visualized in the following workflow.

Protoplast Transfection for CRISPR Editing

For genome editing, transfection is performed after protoplast isolation and before the regeneration cycle begins.

• Adjust purified protoplast density to 400,000-600,000 cells/ml in 0.5 M mannitol.
• Transfection: Mix protoplasts with plasmid DNA (e.g., CRISPR/Cas9 construct) and use PEG-mediated transfection for transient expression. This study achieved 40% transfection efficiency using a GFP marker gene [1].
• After transfection, embed the protoplasts in alginate layers on solid media and initiate the five-stage regeneration protocol.

The Scientist's Toolkit: Essential Reagent Solutions

Successful implementation of this protocol requires high-quality reagents and equipment. Key materials are listed below.

Table 2: Essential Research Reagent Solutions for Protoplast Regeneration and Transfection.

Item	Function / Application	Example / Note
Cellulase & Macerozyme	Enzymatic digestion of cell walls for protoplast isolation.	Cellulase Onozuka R10, Macerozyme R10 [1].
Mannitol	Osmotic stabilizer in enzyme, plasmolysis, and culture media.	Maintains protoplast integrity [1].
Plant Growth Regulators	Directing cell fate (division, callus, shoot formation).	Auxins (2,4-D, NAA), Cytokinins (BAP), Gibberellin (GA3). Quality is critical [1] [5].
Alginate Solution	For embedding protoplasts, providing a supportive matrix.	2.8% sodium alginate in 0.4 M mannitol [1].
PEG Solution	Facilitates the delivery of CRISPR constructs into protoplasts.	Used for PEG-mediated transfection [1].
Culture Media (Base)	Provides essential nutrients and vitamins.	Murashige and Skoog (MS) salts are standard [1] [4].
Agar	Gelling agent for solid culture media.	Provides physical support for growing tissues [1] [5].

Integration in CRISPR Research: Beyond the Protocol

Addressing the Regeneration Bottleneck

The optimized protocol directly addresses the tissue culture bottleneck that slows CRISPR application in many crops [2]. By providing a clear, stage-specific roadmap for PGR manipulation, it increases the odds of successfully regenerating plants from edited protoplasts. This is crucial for producing "DNA-free" edited plants, where the CRISPR machinery is delivered transiently without integrating foreign DNA into the genome, potentially simplifying regulatory approval [1].

Future Perspectives: Automation and Tissue Culture-Independent Methods

The future of plant tissue culture for CRISPR research lies in integrating advanced technologies and developing novel delivery methods.

• Automation and AI: Robotic systems (e.g., RoBoCut) and AI-driven machine vision are being deployed to automate repetitive tasks like cutting and transferring cultures, reducing labor costs by up to 86% and improving consistency [3]. Artificial Neural Networks (ANNs) can predict optimal media formulations, drastically reducing protocol development time [3].
• Tissue Culture-Independent (TCI) Transformation: Emerging techniques aim to deliver CRISPR components directly into plant meristems in planta, completely bypassing the need for tissue culture. A 2025 study used a modified plant virus to deliver CRISPR components, creating heritable edits without tissue culture, a promising solution for recalcitrant species [3].

The convergence of these technologies is paving the way for autonomous bio-factories, integrating AI, automation, and CRISPR into a seamless "Design-Build-Test-Learn" cycle for accelerated crop improvement [3].

The five-stage protoplast regeneration protocol for Brassica carinata exemplifies how a meticulously optimized tissue culture system can serve as a highly efficient gateway to regenerating CRISPR-edited plants. By mastering the precise control of plant growth regulators across distinct developmental stages, researchers can achieve high regeneration and transfection efficiencies, turning a persistent bottleneck into a robust pipeline. As the field advances, the integration of such refined protocols with automation, AI, and novel delivery systems will undoubtedly unlock the full potential of CRISPR-based crop improvement.

Application Notes

The CRISPR/Cas9 system has revolutionized plant genome engineering, enabling precise modifications for functional genomics and trait improvement. A key application in plant biotechnology is the development of marker-free transgenic plants, addressing regulatory and biosafety concerns associated with traditional genetically modified crops.

Efficient Excision of Selectable Marker Genes

A primary application of CRISPR/Cas9 in plant tissue culture is the precise elimination of selectable marker genes (SMGs) from established transgenic plant lines. SMGs are essential for initial selection of transformed tissue but raise significant biosafety concerns and regulatory hurdles for commercial crop release. A multiplex CRISPR/Cas9 strategy successfully excised a DsRED SMG cassette from transgenic tobacco plants. The protocol achieved approximately 10% excision efficiency in the T0 generation, with subsequent segregation in the T1 generation yielding transgene-free plants lacking both the SMG and CRISPR machinery. These edited plants displayed normal growth, flowering, and seed production, confirming the non-disruptive nature of this method for plant development and fertility [6].

Multiplex Editing for Complex Trait Engineering

Multiplex CRISPR/Cas9 systems, which employ multiple guide RNAs (gRNAs) simultaneously, are powerful tools for manipulating complex traits. This approach is highly effective for generating large genomic deletions or knocking out multiple genes within a family to overcome functional redundancy. For instance, researchers have developed genome-wide multi-targeted CRISPR libraries in tomatoes comprising 15,804 unique sgRNAs designed to target multiple genes within the same families. This large-scale effort generated approximately 1,300 independent lines with distinct phenotypes affecting fruit development, flavour, and disease resistance. This multi-targeted strategy offers enhanced efficiency for large-scale crop improvement compared to traditional single-gene editing [7].

Table 1: Key Quantitative Data from CRISPR/Cas9 Applications in Plants

Application / Organism	Efficiency / Outcome	Key Parameters	Reference
SMG Excision (Tobacco)	~10% excision efficiency (T0)	4 gRNAs, Cas9; Normal plant development	[6]
Multiplex Editing (Tomato)	~1,300 independent lines	15,804 sgRNAs; Phenotypes in fruit and disease	[7]
Platycodon grandiflorus Editing	16.70% editing efficiency	Target: endogenous chr2.2745 gene	[7]
Ribonucleoprotein Delivery (Carrot)	17.3% and 6.5% editing rates	Protoplast RNP delivery; Transgene-free plants	[7]

Experimental Protocols

Agrobacterium-mediated Transformation and Regeneration of Tomato for CRISPR/Cas9 Gene Editing

This protocol provides a detailed methodology for generating transgene-free, edited tomato plants using CRISPR/Cas9, a process taking 6–12 months from transformation to a homozygous edited plant [8] [9].

Key Reagents and Biological Materials

• Vector System: Golden Gate-compatible modules (e.g., pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST for Cas9, pICSL01009::AtU6p for sgRNAs) [8] [9].
• Agrobacterium tumefaciens Strain: GV3101 [8] [9].
• Plant Material: Solanum lycopersicum cv. MoneyMaker Cf-0 [8] [9].
• Culture Media: See Table 2 for compositions.

Table 2: Culture Media for Tomato Transformation and Regeneration

Medium Name	Base Composition	Key Additives (Post-Sterilization)	Purpose
½ MS	2.15 g/L MS + Gamborg B5 vitamins, 10 g/L sucrose, 8 g/L agar	(None)	Seed germination
Cocultivation Medium I (CIM I)	4.3 g/L MS + Gamborg B5 vitamins, 30 g/L sucrose, 5.2 g/L Phytoagar	1 mg/L thiamine HCl, 1 mg/L 2,4-D, 0.2 mg/L kinetin	Callus induction
Cocultivation Medium II (CIM II)	Same as CIM I	Same as CIM I, plus 200 μM acetosyringone	Agrobacterium cocultivation
Shoot Induction Medium I (SIM I)	Same as CIM I	1 mg/L thiamine HCl, 2 mg/L trans-Zeatin, 100 mg/L kanamycin, 250 mg/L timentin	Shoot induction under selection
Shoot Induction Medium II (SIM II)	Same as CIM I	1 mg/L thiamine HCl, 1 mg/L trans-Zeatin, 100 mg/L kanamycin, 250 mg/L timentin, 0.1 mg/L IAA	Shoot elongation
Root Induction Medium (RIM)	Same as CIM I	50 mg/L kanamycin, 250 mg/L timentin, 1 mg/L IAA	Rooting of regenerated shoots

Step-by-Step Procedure

• sgRNA Cloning and Vector Assembly: Design two sgRNAs targeting the first exon downstream of the start codon of your gene of interest. Clone the sgRNA expression cassettes into a binary vector containing a plant-optimized Cas9 and a kanamycin selectable marker for plants using Golden Gate assembly [8] [9].
• Transformation into Agrobacterium: Introduce the assembled binary vector into A. tumefaciens strain GV3101 using standard transformation techniques [8] [9].
• Tomato Transformation:
  • Sterilization & Explant Preparation: Surface-sterilize tomato seeds and germinate on ½ MS medium. Use cotyledons from 7-10 day old seedlings as explants [8] [9].
  • Cocultivation: Pre-culture explants on CIM I medium for 2 days. Inoculate explants with an Agrobacterium suspension (OD₆₀₀ ~0.6-0.8) for 20-30 minutes, then cocultivate on CIM II medium in the dark for 2 days [8] [9].
  • Selection and Regeneration: Transfer explants to SIM I medium to induce shoot formation under selection. Subculture every 2 weeks to fresh SIM I medium. Once shoots begin to elongate, transfer to SIM II medium to promote further growth [8] [9].
  • Rooting: Excise developed shoots and transfer to RIM medium to induce root formation [8] [9].
• Screening and Genotyping:
  • Extract genomic DNA from regenerated plantlets.
  • Perform PCR amplification of the target region and sequence the products to identify indel mutations. The use of two sgRNAs facilitates the detection of larger deletions [8].
• Segregation for Transgene-Free Plants:
  • Self-pollinate primary (T0) edited plants and collect seeds (T1 generation).
  • Germinate T1 seeds on antibiotic selection medium to identify plants that have lost the Cas9/sgRNA T-DNA.
  • Genotype the resulting, transgene-free plants to identify homozygous edited lines [8] [9].

Protocol for CRISPR/Cas9-Mediated Removal of Selection Markers

This protocol is designed to remove selectable marker genes from previously generated transgenic plants [6].

Key Reagents and Biological Materials

• Plant Material: Established transgenic tobacco line containing the SMG (e.g., DsRED) and gene of interest (GOI).
• CRISPR Vector: A vector containing Cas9 and four gRNAs designed to target flanking regions of the SMG cassette.

Step-by-Step Procedure

• gRNA Design and Vector Construction: Design four gRNAs targeting sequences immediately upstream and downstream of the SMG cassette. Clone these into a CRISPR/Cas9 binary vector [6].
• Re-transformation: Harvest leaf discs from the SMG-containing transgenic plant and re-transform them with the multiplex CRISPR vector via Agrobacterium-mediated transformation [6].
• Regeneration and Primary Screening: Regenerate shoots on selection medium appropriate for the CRISPR vector. Screen regenerated shoots for the loss of the SMG (e.g., loss of red fluorescence for DsRED). Approximately 20% of shoots may show loss of the marker [6].
• Molecular Confirmation:
  • Perform PCR with primers flanking the SMG cassette. Successful excision will result in a smaller amplicon.
  • Sequence the PCR products to confirm precise deletion and identify any small indels at the gRNA target sites.
  • Use quantitative real-time PCR (qPCR) to confirm the absence of SMG transcripts [6].
• Recovery of Marker-Free and Cas9-Free Plants: Self-pollinate T0 plants with successful SMG excision. Screen the T1 progeny for segregation of the CRISPR T-DNA to recover plants that are devoid of both the SMG and the CRISPR/Cas9 machinery [6].

Visualization

The following workflow diagrams were generated using Graphviz DOT language, adhering to the specified color palette and contrast rules.

CRISPR Plant Genome Editing Workflow

Multiplex Editing for Marker Excision

The Scientist's Toolkit

Table 3: Essential Research Reagents for CRISPR/Cas9 Plant Experiments

Reagent / Material	Function / Application	Specific Examples / Notes
CRISPR Vector System	Delivers Cas9 and gRNA(s) into plant cells.	Golden Gate-compatible modules (e.g., pICH47742 for Cas9, pICSL01009 for sgRNA); Vectors with different promoters and markers for flexibility [8] [9].
Agrobacterium Strain	Mediates the transfer of T-DNA containing the CRISPR machinery into the plant genome.	GV3101, LBA4404. Choice of strain can affect transformation efficiency [6] [8].
Plant Selectable Markers	Selects for plant cells that have successfully integrated the T-DNA.	Kanamycin (NPTII gene), Hygromycin. Critical for initial transformation and selection of edited events [6] [8].
Culture Media & Hormones	Supports plant cell growth, callus induction, and shoot/root regeneration.	MS basal medium; Auxins (2,4-D, IAA) for callus; Cytokinins (Zeatin, Kinetin) for shoot formation [8] [9].
Detection & Validation Kits	Confirms the presence and nature of genetic edits.	Plant genomic DNA extraction kits; PCR reagents; Sanger sequencing; qPCR for expression analysis (e.g., to confirm SMG removal) [6] [10].
Antibiotics (Bacterial)	Maintains plasmid integrity in bacterial and Agrobacterium cultures.	Kanamycin, Rifampicin, Gentamicin. Used in liquid and solid media for bacterial selection [8] [9].

The advent of programmable genome editing technologies has revolutionized molecular biology and agricultural biotechnology. Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) pioneered the field of precise genetic alterations by enabling targeted double-strand breaks (DSBs) in DNA [11]. These protein-based systems demonstrated the feasibility of moving beyond random mutagenesis to intentional genome modification. However, the emergence of the CRISPR-Cas system, particularly CRISPR-Cas9, has represented a paradigm shift due to its simplicity, efficiency, and versatility [12]. For plant research, where genetic manipulation has traditionally been hampered by long breeding cycles and complex regeneration protocols, CRISPR technology offers unprecedented opportunities to accelerate crop improvement and functional genomics studies within the context of plant tissue culture and regeneration systems [13] [2].

This application note provides a comparative analysis of these three major editing platforms, with a specific focus on their utility in plant biotechnology. We detail experimental protocols for implementing CRISPR in plant systems, visualize key workflows, and provide a reagent toolkit to facilitate adoption by researchers engaged in the development of CRISPR-edited plants.

Comparative Analysis of Editing Platforms

Mechanism of Action

The fundamental mechanism by which programmable nucleases operate involves creating a double-strand break (DSB) at a specific genomic location, which is then repaired by the cell's endogenous DNA repair machinery—either through error-prone Non-Homologous End Joining (NHEJ) or precise Homology-Directed Repair (HDR) [12]. Despite this shared principle, the molecular architectures and targeting mechanisms of ZFNs, TALENs, and CRISPR-Cas systems differ significantly.

• Zinc Finger Nucleases (ZFNs): ZFNs are chimeric proteins comprising a DNA-binding domain and a cleavage domain. The DNA-binding domain consists of multiple zinc finger motifs, each recognizing a specific 3-base pair DNA triplet [14] [15]. The cleavage domain is provided by the FokI restriction endonuclease, which requires dimerization to become active [15]. Consequently, a pair of ZFNs must be designed to bind opposite DNA strands with a spacer between them, allowing the two FokI domains to dimerize and cleave the DNA within the spacer region [11].

• Transcription Activator-Like Effector Nucleases (TALENs): Similar to ZFNs, TALENs also fuse a DNA-binding domain to the FokI nuclease domain [14]. However, the DNA-binding domain is derived from TALE proteins of plant-pathogenic bacteria. This domain is built from a series of 33-35 amino acid repeats, each recognizing a single nucleotide [15]. The nucleotide specificity is determined by two hypervariable amino acids at positions 12 and 13, known as the Repeat Variable Diresidue (RVD) [12]. Like ZFNs, TALENs function as pairs, binding to opposing DNA strands and cleaving the spacer DNA via FokI dimerization [11].

• CRISPR-Cas Systems: The CRISPR-Cas system functions fundamentally differently. It is an RNA-guided system where a guide RNA (gRNA) directs the Cas nuclease (e.g., Cas9) to a complementary DNA sequence [11] [12]. The gRNA is a synthetic fusion of crRNA (which provides the target-specific sequence) and tracrRNA (which serves as a scaffold for Cas9 binding) [14]. Cas9 cleaves the DNA upon recognizing a short Protospacer Adjacent Motif (PAM) sequence adjacent to the target site [14]. For the commonly used Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence is 5'-NGG-3' [14]. This RNA-DNA recognition mechanism eliminates the need for complex protein engineering for each new target.

Figure 1: Comparative mechanisms of ZFNs, TALENs, and CRISPR-Cas9. ZFNs and TALENs rely on protein-DNA recognition and require FokI dimerization for cleavage. CRISPR-Cas9 uses a guide RNA for DNA recognition and a single Cas9 nuclease for cleavage, requiring only a PAM site adjacent to the target sequence.

Performance and Practicality Comparison

The distinct mechanisms of these three platforms translate directly into differences in performance, practicality, and suitability for various applications, particularly in plant research.

Table 1: Comprehensive Comparison of Gene Editing Platforms

Feature	CRISPR-Cas9	TALENs	ZFNs
Targeting Mechanism	RNA-DNA base pairing [14]	Protein-DNA recognition [15]	Protein-DNA recognition [15]
Target Specificity	20-nucleotide guide RNA + PAM (e.g., NGG) [14]	12-20 bp per monomer, requires 5'-T [12]	9-18 bp per monomer, 3 bp/finger [14]
Nuclease Component	Cas9 protein	FokI dimer [11]	FokI dimer [11]
Ease of Design	Very simple (within a week) [12]	Complex (∼1 month) [12]	Complex (1–6 months) [11] [12]
Cost	Low [11] [12]	Medium to High [12] [14]	High [11] [12]
Multiplexing Capacity	High (multiple gRNAs) [11]	Limited [11]	Very Limited [11]
Typical Efficiency	High to very high [16]	Moderate to High [15]	Moderate [15]
Off-Target Effects	Moderate to High (subject to off-target effects) [11] [17]	Low (less prone to off-target effects) [17]	Low to Medium [14]
Delivery Size	~4.2 kb (SpCas9) + ~0.1 kb (gRNA)	~3 kb per monomer [15]	~1 kb per monomer [15]
Key Advantage	Simplicity, cost, multiplexing [11]	High specificity, flexible targeting [17]	High precision, established history [17]
Main Disadvantage	Off-target effects, PAM dependency [17]	Large size, complex design [11]	Complex design, high cost [11]

For plant scientists, the implications of this comparison are profound. The simplicity and low cost of CRISPR design make it possible to target multiple genes simultaneously (multiplexing), a crucial feature for studying gene networks and manipulating complex polygenic traits in crops [11] [14]. Furthermore, the rapid design cycle allows for high-throughput functional genomics screens, such as CRISPR knockout libraries, to identify genes involved in stress tolerance or yield [11]. While TALENs and ZFNs can offer high specificity, their technical complexity and cost often render them impractical for large-scale projects in all but the most specialized applications [2].

CRISPR Workflow for Plant Tissue Culture

Implementing CRISPR in plant systems involves a multi-stage process that integrates molecular biology with plant tissue culture techniques. The following protocol outlines the key steps from target selection to the regeneration of edited plants, highlighting critical considerations for success.

Experimental Protocol

Protocol: CRISPR-Cas Mediated Gene Editing in Plants via Tissue Culture

I. Design and Cloning Phase

• Target Selection and gRNA Design:

  • Identify a 20-nucleotide target sequence adjacent to a 5'-NGG PAM sequence in your gene of interest [14].
  • Use online tools (e.g., CRISPR-P, CHOPCHOP) to assess target specificity and potential off-target sites in the plant's genome.
  • Design and synthesize two oligonucleotides corresponding to your chosen target.

• Vector Construction:

  • Select an appropriate plant CRISPR binary vector (e.g., pRGEB, pHEE401).
  • Anneal and phosphorylate the oligonucleotides, then ligate them into the BsaI site of the gRNA expression cassette [18].
  • Transform the ligation product into competent E. coli cells, screen for positive clones, and sequence-verify the final construct.

II. Delivery and Regeneration Phase

• Plant Transformation:

  • For Agrobacterium-mediated transformation: Introduce the binary vector into a disarmed Agrobacterium tumefaciens strain (e.g., LBA4404, GV3101).
  • Prepare explants (e.g., leaf disks, embryogenic calli, shoot apices) from sterile seedlings or in vitro plants.
  • Co-cultivate explants with the Agrobacterium suspension for 2-3 days.
  • Transfer explants to selection media containing antibiotics (e.g., kanamycin, hygromycin) to eliminate non-transformed tissue and induce callus formation.

• Tissue Culture and Regeneration:

  • Maintain and subculture developing calli on fresh selection media.
  • Transfer putative transgenic calli to regeneration media supplemented with cytokinins (e.g., BAP) and auxins (e.g., NAA) to promote shoot organogenesis.
  • Once shoots develop, transfer them to rooting media containing auxins to induce root formation.
  • Acclimate regenerated plantlets to greenhouse conditions.

III. Analysis and Validation Phase

• Molecular Characterization:
  • Extract genomic DNA from regenerated plantlets.
  • Perform PCR amplification of the target region.
  • Analyze editing efficiency using restriction fragment length polymorphism (RFLP) assays if the edit disrupts a restriction site, or by sequencing the PCR products to characterize the exact mutations (indels) introduced by NHEJ.

Figure 2: CRISPR workflow for plant gene editing. The process integrates molecular cloning, delivery of editing components into plant cells, tissue culture regeneration under selection, and final molecular validation of edits. Key bottlenecks like recalcitrant regeneration are addressed by tissue culture-independent (TCI) methods.

Advanced Strategies: Overcoming the Tissue Culture Bottleneck

A significant challenge in plant gene editing is that many crops are recalcitrant to regeneration from tissue culture [13] [2]. This creates a bottleneck where edited cells cannot be regenerated into whole plants. To address this, Tissue Culture-Independent (TCI) methods are being developed [13] [2].

• Virus-Induced Genome Editing (VIGE): Engineered plant viruses (e.g., Tobacco Rattle Virus) are used to deliver CRISPR components directly into the plant's meristematic cells, bypassing the need for in vitro culture. A 2025 study demonstrated using a common plant virus to deliver a miniature CRISPR system, creating heritable edits without foreign DNA integration [13].
• Grafting: A transformed, editable plant (the "stock") is grafted onto a non-transformed rootstock. The mobile gRNA or Cas9 protein is theorized to move across the graft junction to edit the rootstock cells.
• Nanoparticle Delivery: CRISPR reagents are encapsulated in biodegradable nanoparticles and delivered directly to plant meristems.

These advanced delivery strategies, particularly the use of ribonucleoprotein (RNP) complexes (pre-assembled Cas9 protein and gRNA), can also help generate transgene-free edited plants, as the RNP complex degges naturally after editing, leaving no foreign DNA behind [19]. This simplifies regulatory approval and public acceptance.

The Scientist's Toolkit: Research Reagent Solutions

Successful implementation of CRISPR-based plant genome editing requires a suite of reliable reagents and materials. The following table details essential components and their functions.

Table 2: Essential Reagents and Materials for CRISPR Plant Research

Reagent/Material	Function/Description	Key Considerations
CRISPR Vector System	A binary plasmid for plant transformation containing Cas9 and gRNA expression cassettes [18].	Choose species-optimized systems (e.g., with plant-promoters like Ubi, 35S). Modular systems (e.g., Golden Gate) simplify gRNA cloning.
Cas9 Nuclease	The effector protein that creates the double-strand break.	SpCas9 is standard; consider smaller variants (SaCas9) or altered PAM specificity (xCas9, SpCas9-NG) for expanded targeting [12].
Guide RNA (gRNA)	Synthetic RNA that directs Cas9 to the target DNA sequence.	Can be expressed from a U6 or U3 pol III promoter in the vector. Specificity is critical to minimize off-target effects.
Plant Binary Vector	A T-DNA based plasmid used for Agrobacterium-mediated transformation.	Must contain left and right border sequences, plant selection marker (e.g., hptII, bar), and bacterial selection marker.
Agrobacterium Strain	A disarmed plant pathogen used as a vector for DNA delivery (e.g., LBA4404, GV3101) [18].	Strain choice can significantly impact transformation efficiency in different plant species.
Tissue Culture Media	Nutrient media supporting plant cell growth and regeneration (e.g., MS, B5 media).	Must be supplemented with appropriate plant growth regulators (auxins, cytokinins) and selection agents (antibiotics/herbicides).
Selection Agents	Antibiotics (e.g., Kanamycin, Hygromycin) or herbicides (e.g., Bialaphos) to select transformed tissues.	The choice depends on the resistance marker in the CRISPR vector. Dose must be optimized for each species.
Plant Growth Regulators	Hormones like auxins (2,4-D, NAA) and cytokinins (BAP, Zeatin) to direct callus formation and shoot/root development.	The ratio and concentration are species-specific and critical for efficient regeneration.

The comparative analysis presented in this application note unequivocally demonstrates why CRISPR-Cas has become the predominant genome editing technology in plant research. Its simplicity of design, cost-effectiveness, and unparalleled multiplexing capacity offer a clear and decisive advantage over older protein-based platforms like ZFNs and TALENs [11] [14]. This technological superiority is accelerating fundamental research and the development of improved crop varieties with enhanced yield, quality, and resilience [18].

However, the full potential of CRISPR in plant biotechnology can only be realized by integrating it with robust plant tissue culture and regeneration systems. The persistent challenge of regeneration recalcitrance in many key crops underscores the critical importance of ongoing research into tissue culture-independent delivery methods [2]. As these protocols become more refined and accessible, CRISPR is poised to fully democratize precise genome manipulation, empowering researchers worldwide to contribute to a new era of precision plant breeding and sustainable agriculture.

For researchers aiming to develop improved crops through CRISPR-based genome editing, the recovery of viable, genetically stable plants is a fundamental step. This process relies critically on plant tissue culture, a set of techniques that enables the regeneration of whole plants from single cells or tissues that have undergone genetic modification. While CRISPR/Cas systems provide the means for precise genomic alterations, tissue culture offers the essential pathway for these edits to be amplified and transmitted to subsequent generations.

The synergy between these technologies is particularly vital for addressing a major bottleneck in plant biotechnology: the recalcitrance of many crop species to genetic transformation and regeneration [2]. Even with efficient editing tools, the inability to regenerate plants from edited cells remains a significant barrier for many crops, especially perennials, woody species, and clonally propagated plants [20]. This protocol details established and emerging tissue culture methods that enable researchers to overcome these challenges and successfully recover CRISPR-edited plants.

Key Tissue Culture Platforms for CRISPR Plant Recovery

Established Regeneration Systems

The table below summarizes three primary tissue culture approaches used for recovering CRISPR-edited plants, along with their applications in recent research.

Table 1: Tissue Culture Platforms for Recovering CRISPR-Edited Plants

Tissue Culture Method	Key Applications in CRISPR Plant Research	Representative Species	Key Advantages
Nodal Culture & Meristem-Based Regeneration [20]	Regeneration of recalcitrant horticultural crops; Reduces issues with desiccation and contamination.	Coffea arabica, Citrus limon, Garcinia mangostana [20]	Utilizes pre-existing meristems; High genetic stability; Bypasses somaclonal variation.
Protoplast Regeneration [21]	DNA-free editing with RNP complexes; Ensures uniform edits from single cells.	Temperate japonica rice (Oryza sativa L.) [21]	Avoids chimerism; No foreign DNA integration; High editing uniformity.
Synthetic Regeneration Systems [22]	Bypasses traditional tissue culture; Direct shoot generation from wounded tissue.	Tobacco, Tomato, Soybean [22]	Culture-free; Faster regeneration; Genotype-independent potential.

Experimental Protocols

Protocol 1: Nodal Culture for Recalcitrant Crops

This protocol is adapted from methods successfully used for regenerating challenging horticultural species [20].

• Explant Selection and Sterilization:

  • Collect immature nodal segments (1-2 cm in length) from healthy donor plants.
  • Cleanse segments with a liquid detergent (e.g., 0.1% Tween 20) for 20 minutes, followed by thorough rinsing with distilled water.
  • Surface-sterilize by immersion in 70% ethanol for 5 minutes, followed by treatment with 1% sodium hypochlorite for 20 minutes.
  • Rinse explants 4-5 times with sterile distilled water to remove all traces of disinfectants.

• Culture Initiation and Shoot Regeneration:

  • Inoculate sterilized nodal explants onto solid Murashige and Skoog (MS) or Driver-Kuniyuki (DKW) medium.
  • Supplement the medium with a combination of plant growth regulators:
    • Auxin (e.g., 0.01–2 mg/L)
    • Cytokinin (e.g., 0.4–4 mg/L)
  • Maintain cultures at 25 ± 2°C under a 16-hour light/8-hour dark photoperiod.
  • Shoot regeneration typically occurs within 4-8 weeks.

• Root Induction and Acclimatization:

  • Transfer regenerated shoots (2-3 cm in length) to root induction media, such as half-strength Woody Plant Medium (WPM) supplemented with a lower concentration of auxin (0.1–2 mg/L).
  • Once a healthy root system develops (approximately 4 weeks), acclimatize plantlets in a controlled environment with high relative humidity (>85%) using a peat:perlite (2:1) mixture.
  • Gradually transfer hardened plants to greenhouse conditions over 15-30 days.

Diagram: Key Molecular Regulators in Nodal Culture Regeneration

Protocol 2: Protoplast-Based Regeneration and Editing

This protocol outlines a method for achieving non-transgenic, CRISPR-edited plants in rice, adaptable to other species [21].

• Induction of Embryogenic Callus:

  • Culture mature seeds on 2N6 medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) to induce somatic embryogenesis.
  • Subculture friable, pale yellow embryogenic calli every two weeks to maintain viability.

• Protoplast Isolation:

  • Digest 500 mg of embryogenic callus (approximately 2 months old) in an enzymatic solution containing:
    • 1.5% (w/v) Cellulase Onozuka R-10
    • 0.75% (w/v) Macerozyme R-10
    • Dissolved in 0.6 M AA medium
  • Incubate in the dark at 28°C with gentle shaking (40 rpm) for 18-20 hours.
  • Purify released protoplasts by filtering through a mesh and washing with W5 solution. Expected viability should range from 70-99%.

• Transfection and Genome Editing:

  • Transfer purified protoplasts to a MMg solution.
  • For CRISPR editing, transfert protoplasts with PEG-mediated delivery of preassembled Cas9-gRNA ribonucleoprotein (RNP) complexes.
  • Incubate transfected protoplasts in the dark at 28°C for 48 hours for transient expression.

• Regeneration of Whole Plants:

  • Culture transfected protoplasts using alginate bead embedding in 2N6 medium, cocultured with feeder extracts.
  • Embryogenic callus formation typically occurs within 35 days.
  • Transfer developed calli to N6R and N6F media for shoot and root regeneration.
  • Acclimatize regenerated plantlets in a greenhouse within three months.

Diagram: Workflow for Protoplast-Based CRISPR Plant Regeneration

The Scientist's Toolkit: Essential Research Reagents

Successful implementation of the protocols above requires a suite of specialized reagents and materials. The following table details the key components and their functions in the tissue culture and CRISPR editing pipeline.

Table 2: Essential Research Reagents for Tissue Culture and CRISPR Plant Recovery

Reagent/Material	Function/Application	Specific Examples/Notes
Culture Media [20]	Provides essential nutrients and osmotic environment for growth.	MS, DKW, WPM; Choice depends on plant species.
Plant Growth Regulators [20] [22]	Direct cell fate (division, elongation, differentiation).	Auxins (e.g., 2,4-D), Cytokinins (e.g., BAP); Ratio is critical.
Cell Wall-Degrading Enzymes [21]	Digest cell wall to release protoplasts.	Cellulase Onozuka R-10, Macerozyme R-10.
CRISPR Delivery Vectors [4] [23]	Introduce Cas9 and gRNA into plant cells.	Agrobacterium (e.g., strain EHA105), RNP complexes.
Selection Agents [4]	Select for successfully transformed cells.	Kanamycin (20-70 mg/L); Species-specific concentration required.
Sterilization Agents [20]	Surface sterilize explants to prevent contamination.	Ethanol (70%), Sodium Hypochlorite (0.8-1%).

The integration of advanced tissue culture methods with CRISPR/Cas9 technology is pivotal for translating genomic edits into viable, improved plants. While challenges remain—particularly with recalcitrant species—the continued refinement of regeneration protocols, including nodal culture, protoplast systems, and novel synthetic biology approaches, is steadily expanding the range of editable crops. Mastery of these synergistic techniques empowers researchers to effectively contribute to the development of resilient, high-yielding crop varieties essential for future food security. Future directions will likely involve the increased application of AI and machine learning to optimize culture conditions and predict editing outcomes, further enhancing the efficiency and precision of this powerful combined technology [24].

Current Market and Research Landscape for Plant Biotechnology

The plant biotechnology market is experiencing significant growth, propelled by the urgent global need to enhance food security, develop climate-resilient crops, and promote sustainable agricultural practices. The market is anticipated to grow from $51.73 billion in 2025 to $76.79 billion by 2030, reflecting a compound annual growth rate (CAGR) of 8.2% [25] [26]. Alternative analyses project a slightly higher baseline of $58.4 billion in 2025, rising to $117.7 billion by 2034 at a CAGR of 8.1% [27]. This expansion is primarily driven by the rising adoption of biotech seeds, advancements in gene-editing technologies, and the critical need for crop resilience in the face of climate change [25] [27].

Table 1: Global Plant Biotechnology Market Size and Growth Projections

Market Size (2025)	Projected Market Size	CAGR	Source Year
USD 51.73 billion	USD 76.79 billion by 2030	8.2%	[25] [26]
USD 58.4 billion	USD 117.7 billion by 2034	8.1%	[27]

A key trend disrupting the market is the rapid advancement and application of CRISPR-based genome editing [27]. This technology enables more precise genetic modifications to develop crops with enhanced traits, such as disease resistance, improved nutritional content, and better tolerance to environmental stresses like drought [28]. Furthermore, there is a noticeable shift towards sustainable alternatives, driving growth in the bio-based pesticides and fertilizers segment [25] [27]. The broader agricultural biotechnology market, which includes animal applications, is on a similar trajectory, expected to grow from $126.3 billion in 2024 to $294.63 billion by 2034 [29].

Market Segmentation Analysis

The plant biotechnology market can be segmented by product, technology, crop type, and end-user, each with distinct leaders and growth patterns.

By Product Type

The Biotech Seeds & Traits segment currently dominates the market [25]. This leadership is fueled by the widespread adoption of genetically modified crops with traits like insect resistance and herbicide tolerance, particularly in major row crops such as corn, soybean, and cotton. However, the Synthetic Biology-Enabled Products segment is projected to be the fastest-growing category. This growth is driven by its unprecedented precision in designing novel biological systems to enhance crop traits and improve stress tolerance [27].

By Technology Type

Genetic Engineering holds the largest market share due to its well-established role and extensive commercial application in developing genetically modified crops [25] [27]. Meanwhile, Genome Editing is a rapidly evolving field within the industry. The global gene editing market alone is projected to surpass $13 billion by 2025, with a remarkable CAGR of 17.2% [30].

By Crop Type and End User

Cereals & Grains lead the crop type segment, a status owed to their essential role in global food security and the high adoption of biotech traits in crops like corn and rice [25]. In terms of end-users, Seed Companies are the most dominant, as they are the primary entities leveraging biotechnological advancements to develop and commercialize improved seeds [25].

Table 2: Plant Biotechnology Market Segmentation (2025-2030)

Segmentation	Dominant Segment	Fastest-Growing Segment	Key Drivers
By Product Type	Biotech Seeds & Traits	Synthetic Biology-Enabled Products	Demand for high-yield, climate-resilient crops; precision of synbio [25] [27]
By Technology	Genetic Engineering	Genome Editing (e.g., CRISPR)	Precision, cost-effectiveness, ability to avoid GMO regulations [25] [30]
By Crop Type	Cereals & Grains	Information Not Specified	Essential role in global food security [25]
By End User	Seed Companies	Information Not Specified	Integration of biotech into breeding and commercial seed lines [25]

Regional Landscape

North America is the current market leader, accounting for approximately 45% of global biotech crop acreage [25]. However, the Asia-Pacific region is a major hub and is expected to see significant growth, driven by rapid population increase, strong government support for agricultural biotechnology, and extensive agricultural infrastructure [27] [29]. Europe maintains a strong position characterized by scientific innovation and robust regulatory frameworks, with leading research institutions such as the Max Planck Institute in Germany and Wageningen University in the Netherlands [26].

Application Notes: A Pipeline for CRISPR/Cas9 Genome Editing in Plants

The following protocol provides a detailed, step-by-step pipeline for implementing CRISPR/Cas9 genome editing in plants, from in silico design to the regeneration of edited plants. This workflow is specifically designed for functional gene validation and trait improvement within a plant tissue culture context [31].

Protocol: Step-by-Step Genome Editing Workflow

Objective: To establish a robust pipeline for generating genome-edited plants using CRISPR/Cas9 technology for functional gene validation and trait improvement [31].

In Silico Sequence Analysis

Purpose: To confirm the target gene structure and identify the precise locations for sgRNA design [31]. Procedure:

• Retrieve Sequences: Obtain the genomic DNA, mRNA, and coding sequence (CDS) for the target gene from species-specific databases (e.g., Phytozome, Gramene) [31].
• Map Gene Structure: Align the transcript and CDS to the genomic sequence to map intron/exon boundaries and identify all splicing variants using multiple sequence alignment software (e.g., MAFFT via Benchling) [31].
• Manual Annotation: Manually verify and annotate the target genomic sequence, paying close attention to the start codon and the structure of all transcript variants. This ensures sgRNAs are designed in conserved exonic regions [31].

Design and Validation of Guide RNAs (sgRNAs)

Purpose: To design and select high-efficiency sgRNAs that minimize off-target effects [31]. Procedure:

• Multi-Tool Design: Input the target genomic sequence into multiple online sgRNA design tools (e.g., CRISPR-P 2.0, CHOPCHOP, CRISPR-PLANT v2) [31].
• Identify Common sgRNAs: Cross-reference the outputs from different tools to identify sgRNAs that are consistently recommended. These "common" sgRNAs often cluster in conserved regions and have a higher probability of success [31].
• Select Final sgRNAs: Prioritize sgRNAs based on:
  • Targeting all transcript variants.
  • High predicted efficiency scores.
  • A low number of predicted off-target sites.
  • For knock-outs, design near the 5' end of the coding sequence to maximize the chance of generating frameshift mutations and truncated proteins [31].
• In Vitro Validation (Recommended): Validate the cleavage efficiency of designed sgRNAs using an in vitro CRISPR/Cas9 ribonucleoprotein (RNP) assay before moving to plant transformation [31].

Primer Design and Sequencing of Target Regions

Purpose: To account for natural sequence variation (SNPs, InDels) in the plant material used for transformation, which is critical for sgRNA efficacy [31]. Procedure:

• Design Flanking Primers: Design primers that amplify a 500-1200 bp region surrounding the sgRNA target site using tools like NCBI Primer-BLAST [31].
• Sequence the Target Locus: Amplify the target region from the genotype to be transformed using high-fidelity PCR. Clone the PCR product (e.g., using TOPO TA cloning) and perform Sanger sequencing on multiple clones to determine the exact haplotype(s) present [31].
• Verify sgRNA Compatibility: Ensure the designed sgRNA sequence is a perfect match to the sequenced target region in your specific plant material.

Delivery of Editing Reagents and Plant Transformation

Purpose: To introduce CRISPR/Cas9 reagents into regenerable plant cells. Background: A significant bottleneck in plant gene editing is the delivery of reagents to regenerable cells and the subsequent regeneration of whole plants, a process often reliant on tissue culture [32]. The choice of cargo and vehicle is crucial. Procedure:

• Cargo Options:
  • DNA Vector: Stable expression of Cas9 and sgRNA(s); requires segregation in progeny to obtain transgene-free edits [31] [32].
  • RNA: In vitro transcribed mRNA for Cas9 and sgRNA; reduces transgene integration risk [32].
  • Ribonucleoprotein (RNP): Pre-assembled Cas9 protein and sgRNA complex; considered the "cleanest" method as it is transient and leaves no genetic trace, directly generating edited plants [31] [32].
• Delivery Methods:
  • Agrobacterium-mediated transformation: Most common for DNA delivery [33].
  • Biolistics (Particle bombardment): Useful for species recalcitrant to Agrobacterium [33].
  • PEG-mediated transfection: Typically used for protoplast transformation, which can serve as an intermediate validation step [31] [32].
  • Novel Methods: Emerging techniques like viral vectors or nanoparticle delivery are being developed to avoid tissue culture altogether [32].

Regeneration and Selection of Edited Plants

Purpose: To regenerate whole plants from transformed cells and identify editing events. Procedure:

• Tissue Culture: Transfer transformed tissue to regeneration media to induce shoot and root formation. This step is highly species-genotype specific [31] [34].
• Selection: Use appropriate selective agents (e.g., antibiotics, herbicides) if the transformation vector contains a selectable marker gene. For DNA-free editing (RNPs), selection is not possible, making efficient delivery and screening paramount [32].
• Regenerate Plantlets: Transfer developing shoots to rooting media and subsequently acclimatize plantlets to soil conditions [31].

Mutation Detection and Analysis

Purpose: To identify and characterize the nature of edits in regenerated plants. Procedure:

• Genomic DNA Extraction: Extract DNA from regenerated plantlets.
• PCR Amplification: Amplify the target region from the plant's genome.
• Edit Detection:
  • Sanger Sequencing: Sequence the PCR product directly. A messy chromatogram around the cut site indicates a heterogeneous editing event, necessitating cloning and sequencing of individual alleles [31].
  • Next-Generation Sequencing (NGS): For a high-throughput and quantitative assessment of editing efficiency and heterogeneity across a population of plants [31].
  • Restriction Enzyme (RE) Assay: If the edit disrupts a natural restriction site, digestion of the PCR amplicon can quickly indicate successful mutation [31].

The Scientist's Toolkit: Key Research Reagent Solutions

The following table details essential materials and reagents required for establishing a CRISPR/Cas9 genome editing pipeline in plants.

Table 3: Essential Reagents for Plant CRISPR/Cas9 Workflow

Reagent / Material	Function	Examples / Notes
CRISPR/Cas9 System	Engineered nuclease and guide RNA for targeted DNA cleavage.	Cas9 protein for RNP; plasmids expressing Cas9 and sgRNA for DNA-based delivery [31] [32].
sgRNA Design Tools	In silico design and efficiency prediction of guide RNAs.	CRISPR-P 2.0, CHOPCHOP, CRISPR-PLANT v2; using multiple tools is recommended [31].
Plant Material	Source of regenerable explants for transformation.	Immature embryos, cotyledons, or hypocotyls, depending on species [31].
Tissue Culture Media	Support growth, division, and regeneration of plant cells.	Callus induction media, regeneration media, rooting media; composition is species-specific [31] [34].
Transformation Vectors	Delivery of CRISPR machinery into plant cells.	Plasmids for Agrobacterium or biolistics; can include plant-specific promoters and selectable markers [31] [33].
Delivery Reagents	Facilitate entry of editing reagents into plant cells.	Agrobacterium tumefaciens strains; gold/tungsten particles for biolistics; PEG for protoplasts [33] [32].
Selection Agents	Enrich for transformed cells and plants.	Antibiotics (e.g., kanamycin, hygromycin) or herbicides, corresponding to the resistance marker on the vector [32].
DNA Polymerase	Amplification of target loci for sequencing and analysis.	High-fidelity PCR enzymes to minimize amplification errors [31].
Sequencing Services	Confirmation of edits and analysis of off-target effects.	Sanger sequencing for initial screening; NGS for deep characterization of edits [31].

Key Workflow Relationships and Bottlenecks

The entire process of developing a genome-edited plant, from gene discovery to a commercial product, is complex and faces several challenges. The diagram below illustrates the key stages and major bottlenecks, particularly highlighting the crucial role of tissue culture and regeneration.

The overall success probability of obtaining an edited plant is a product of the success rates of each independent step: P(success) = P(deliver) x P(cut) x P(repair) x P(regenerate) x P(identify) [32]. As shown in the diagram, tissue culture and regeneration remain a primary bottleneck, as many agronomically important species and elite cultivars are recalcitrant to these processes [34] [32]. Furthermore, the delivery of editing reagents past the plant cell wall is a fundamental challenge, driving research into novel methods like nanoparticle and viral vector delivery [32]. Finally, even after a successful edit is achieved, the regulatory pathway for gene-edited crops can be lengthy and uncertain, varying significantly by jurisdiction and acting as a major market restraint [25].

Advanced delivery and regeneration methods for CRISPR-edited plants

Agrobacterium-mediated transformation is a cornerstone technique in plant biotechnology for the stable integration of foreign DNA into plant genomes. Within the broader context of plant tissue culture for CRISPR-edited plant research, this method provides a reliable and efficient pathway for delivering CRISPR-Cas9 constructs into plant cells. The natural DNA transfer mechanism of Agrobacterium tumefaciens facilitates the integration of T-DNA from its tumor-inducing (Ti) plasmid into the plant genome, making it an ideal vector for CRISPR components [35]. This process is particularly crucial for generating heritable genetic modifications, as it allows for the stable incorporation of Cas9 endonuclease and single-guide RNA (sgRNA) sequences, enabling precise targeted genome editing in regenerated plants.

The synergy between Agrobacterium-mediated transformation and CRISPR technology has revolutionized plant genetic engineering, creating unprecedented opportunities for crop improvement. This combination allows researchers to move beyond simple gene knockouts to achieve precise nucleotide substitutions, gene insertions, and multiplexed editing of several genes simultaneously. When framed within a thesis on plant tissue culture, this technology represents a critical bridge between gene discovery and the development of improved crop varieties with enhanced agronomic traits, providing a more efficient alternative to traditional breeding methods [36] [35].

Key Principles and Mechanisms

Molecular Mechanism of T-DNA Transfer

The process of Agrobacterium-mediated transformation begins with the recognition of wounded plant tissues by the bacterium. Phenolic compounds released from damaged plant cells activate the bacterial VirA/VirG two-component system, triggering the expression of other virulence (vir) genes [35] [37]. The VirD1/VirD2 complex then processes the T-DNA from the Ti plasmid, producing a single-stranded T-DNA molecule covalently attached to VirD2 at the 5' end. This T-DNA complex is transported through a Type IV Secretion System (T4SS) into the plant cell, guided by VirE2 proteins that protect the single-stranded DNA [37]. Once inside the plant nucleus, the T-DNA integrates into the plant genome through illegitimate recombination, a process facilitated by plant proteins that recognize the VirD2 and VirE2 components [35].

CRISPR-Cas9 System for Genome Editing

The CRISPR-Cas9 system functions as a highly precise DNA-targeting machinery that can be delivered via Agrobacterium T-DNA. The system consists of two key components: the Cas9 nuclease and a single-guide RNA (sgRNA). The sgRNA directs Cas9 to specific genomic loci complementary to its 20-base spacer sequence and adjacent to a Protospacer Adjacent Motif (PAM—typically 5'-NGG-3' for Streptococcus pyogenes Cas9) [36] [38]. Upon binding, Cas9 creates double-stranded breaks (DSBs) approximately 3 bp upstream of the PAM site. The plant cell then repairs these breaks through either error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) [39] [38]. NHEJ often results in insertions or deletions (indels) that can disrupt gene function, while HDR can facilitate precise gene edits when a repair template is provided.

The following diagram illustrates the workflow for generating CRISPR-edited plants through Agrobacterium-mediated transformation:

Critical Parameters for Optimization

Successful Agrobacterium-mediated transformation depends on numerous factors that influence transformation efficiency and the recovery of CRISPR-edited plants. These parameters must be carefully optimized for each plant species and genotype.

Table 1: Key Optimization Parameters for Agrobacterium-Mediated Transformation

Parameter Category	Specific Factor	Optimal Conditions/Considerations	Impact on Efficiency
Biological Factors	Plant Genotype	Species and cultivar-specific response	Determines regeneration capacity and susceptibility to Agrobacterium
	Explant Type	Meristematic tissues, cotyledons, hypocotyls, embryonic axes	Affects regeneration potential and Agrobacterium accessibility
	Agrobacterium Strain	AGL1, EHA105, LBA4404, K599 [37]	Virulence efficiency and host range specificity
Co-cultivation Conditions	Temperature	23-25°C [40]	Critical for T-DNA transfer and integration
	Duration	2-3 days	Balance between sufficient T-DNA transfer and bacterial overgrowth
	Acetosyringone Concentration	100-200 μM [40]	Induces vir gene expression; enhances T-DNA transfer
Selection System	Selective Agent	Hygromycin, kanamycin, basta	Must be optimized to minimize escapes while allowing transformed cell growth
	Selection Timing	Delayed application (3-7 days post-co-cultivation)	Allows recovery and division of transformed cells before selection
CRISPR-Specific Factors	sgRNA Design	High on-target activity, minimal off-target potential [38]	Determines editing efficiency and specificity
	Cas9 Expression	Constitutive vs. tissue-specific promoters	Affects editing efficiency and potential cytotoxicity
	Delivery Format	Binary vector with Cas9 and sgRNA expression cassettes	Ensures coordinated expression of CRISPR components

Protocol: Agrobacterium-Mediated Transformation of Tomato

This detailed protocol for tomato (Solanum lycopersicum) transformation incorporates critical steps for successful CRISPR construct integration and can be adapted for other dicotyledonous species with appropriate modifications [40] [41].

Materials and Reagents

• Plant Material: Surface-sterilized tomato seeds (cv. Micro-Tom recommended)
• Agrobacterium Strain: AGL1 or EHA105 harboring binary CRISPR vector
• Binary Vector: Contains Cas9, sgRNA expression cassette, and plant selection marker
• Culture Media: LB, MS basal medium, co-cultivation medium, selection medium, regeneration medium, rooting medium
• Antibiotics: Appropriate for bacterial and plant selection (kanamycin, hygromycin)
• Induction Agents: Acetosyringone (100-200 μM)

Step-by-Step Procedure

Day 1: Preparation of Explants

• Surface-sterilize tomato seeds using 70% ethanol (1 min) followed by 1-2% sodium hypochlorite (10-15 min) with gentle agitation.
• Rinse 3-5 times with sterile distilled water.
• Germinate seeds on MS basal medium in Magenta boxes or Petri dishes under a 16-h photoperiod at 25°C.
• After 7-10 days, excise cotyledons from seedlings and cut into 5-7 mm segments, ensuring each segment contains a minimal petiolar region.

Day 2: Agrobacterium Preparation and Inoculation

• Inoculate 5 mL of LB medium containing appropriate antibiotics with Agrobacterium strain harboring the CRISPR binary vector.
• Incubate at 28°C with shaking (200 rpm) for 24-48 hours until OD600 reaches 0.6-1.0.
• Centrifuge bacterial culture at 5000 × g for 10 min and resuspend in liquid MS medium or inoculation medium containing acetosyringone (100-200 μM) to OD600 0.2-0.5.
• Immerse cotyledon explants in the Agrobacterium suspension for 15-30 min with gentle agitation.
• Blot explants dry on sterile filter paper to remove excess bacteria.

Day 2-5: Co-cultivation

• Transfer inoculated explants to co-cultivation medium (solid MS with acetosyringone).
• Incubate in darkness at 23-25°C for 2-3 days [40].
• Critical Note: Low-temperature incubation (23°C) significantly improves recovery of resistant colonies [40].

Day 5-30: Selection and Regeneration

• After co-cultivation, transfer explants to selection medium containing antibiotics (e.g., kanamycin 100 mg/L) to inhibit Agrobacterium growth and select for transformed plant cells, along with timentin or carbenicillin to eliminate residual bacteria.
• Use a "sandwich selection method" where explants are placed between layers of selection medium to improve selection efficiency [40].
• Subculture explants to fresh selection medium every 2 weeks.
• Developing shoots should become visible within 3-4 weeks.

Day 30-60: Shoot Elongation and Rooting

• Excise developing shoots (1-2 cm length) and transfer to shoot elongation medium.
• For rooting, transfer elongated shoots to rooting medium containing lower concentrations of selective antibiotics.
• Maintain cultures under 16-h photoperiod at 25°C.

Day 60-90: Molecular Analysis and Acclimatization

• Extract genomic DNA from putative transgenic plantlets using CTAB method.
• Perform PCR screening using primers specific to the Cas9 gene and/or selection marker.
• For CRISPR-edited lines, sequence the target region to verify mutations.
• Acclimatize rooted plantlets to greenhouse conditions by transferring to sterile soil mix and maintaining high humidity for 1-2 weeks before gradual exposure to normal growth conditions.

Advanced Applications and Recent Innovations

Tissue-Culture-Free Transformation

Recent breakthroughs have enabled Agrobacterium-mediated transformation without the need for extensive tissue culture, dramatically accelerating the production of CRISPR-edited plants. A novel approach developed by Patil and colleagues at Texas Tech University utilizes a synthetic regeneration system that combines two powerful genes: WIND1, which triggers cells near wounds to reprogram themselves, and the isopentenyl transferase (IPT) gene, which produces natural plant hormones that promote new shoot growth [22]. This system successfully generated gene-edited shoots in tobacco, tomatoes, and soybeans with minimal reliance on conventional tissue culture, addressing a major bottleneck in plant biotechnology.

The tissue-culture-free approach follows this general workflow:

Engineered Agrobacterium Strains

Advanced Agrobacterium genome engineering has produced specialized strains with enhanced transformation capabilities and improved biosafety profiles. The INTEGRATE system, a CRISPR-associated transposase technology, enables precise genomic modifications in Agrobacterium strains without introducing double-strand breaks [37]. This system has been used to create auxotrophic strains (e.g., thymidine auxotrophs) that require supplemented media for growth, reducing environmental persistence concerns. These engineered strains address biosafety concerns associated with recombinant DNA technologies while improving transformation efficiency for challenging crop species [37].

Analysis of CRISPR Editing Efficiency

Molecular Validation Techniques

Following transformation and regeneration, comprehensive analysis is required to confirm successful integration of T-DNA and evaluate CRISPR editing efficiency. Multiple molecular techniques provide complementary information:

• PCR Analysis: Confirms presence of T-DNA using primers specific to Cas9, sgRNA, or selection marker genes.
• Southern Blotting: Verifies stable integration and copy number of T-DNA.
• Sanger Sequencing: Identifies specific mutations at target loci.
• Next-Generation Sequencing: Provides comprehensive analysis of on-target and potential off-target edits.

Quantitative Evaluation Methods

Specialized methods have been developed specifically for quantifying CRISPR editing efficiency:

Table 2: Methods for Analyzing CRISPR Editing Efficiency

Method	Principle	Advantages	Limitations	Applications
ICE (Inference of CRISPR Edits)	Uses Sanger sequencing to quantify indels [42]	Cost-effective, quantitative, user-friendly web tool	Limited multiplexing capability	Knockout efficiency analysis, indel characterization
qEva-CRISPR	Multiplex ligation-based probe amplification (MLPA) [39]	Detects all mutation types, quantitative, works with difficult genomic regions	Requires specialized probe design	Simultaneous analysis of multiple targets, detecting large deletions
T7 Endonuclease I Assay	Mismatch cleavage in heteroduplex DNA	Simple, no specialized equipment needed	Cannot detect homozygous mutations, misses some edits	Initial screening of editing efficiency
Restriction Fragment Length Polymorphism	Loss of restriction site due to editing	Simple protocol, cost-effective	Only works if edit alters restriction site	Efficiency assessment when suitable restriction site exists

The ICE tool, developed by Synthego, is particularly valuable for researchers as it calculates editing efficiency (Indel Percentage), model fit (R² score), knockout score (proportion of frameshift or 21+ bp indels), and knock-in score (proportion of sequences with desired knock-in edit) from standard Sanger sequencing data [42]. This method provides next-generation sequencing-quality analysis at a fraction of the cost, making it accessible for most research laboratories.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for Agrobacterium-Mediated CRISPR Transformation

Reagent/Category	Specific Examples	Function/Application	Considerations
Agrobacterium Strains	AGL1, EHA105, LBA4404, K599 [35] [37]	T-DNA delivery to plant cells	Strain selection affects host range and efficiency
Binary Vectors	pX330-U6, lentiCRISPRv2, pCambia series [38]	Carry CRISPR components in T-DNA	Choice of promoters affects Cas9/sgRNA expression
Selection Agents	Hygromycin, Kanamycin, Basta	Selection of transformed plant tissues	Concentration must be optimized for each species
Vir Gene Inducers	Acetosyringone [40]	Enhances T-DNA transfer efficiency	Critical for recalcitrant species
CRISPR Components	Cas9 nucleases, sgRNAs	Targeted DNA cleavage	Modified Cas9 variants can improve specificity
Edit Verification Tools	ICE, qEva-CRISPR, TIDE [39] [42]	Quantify editing efficiency and characterize mutations	Choice depends on required sensitivity and throughput

Agrobacterium-mediated transformation for stable integration of CRISPR constructs represents a powerful methodology that continues to evolve with advancements in synthetic biology and genome engineering. The protocols and parameters outlined in this application note provide researchers with a robust framework for implementing this technology within plant tissue culture systems. Recent innovations, particularly tissue-culture-free transformation methods and engineered Agrobacterium strains, are addressing longstanding challenges in plant genetic engineering, opening new possibilities for crop improvement. As these technologies mature, they promise to accelerate the development of CRISPR-edited plants with enhanced agronomic traits, contributing to global food security and sustainable agricultural practices.

Protoplast Transformation and Ribonucleoprotein (RNP) Delivery for Transgene-Free Editing

The development of transgene-free genome editing systems represents a pivotal advancement in plant biotechnology, addressing regulatory concerns and technical limitations associated with traditional genetic modification. Central to this advancement is the combination of protoplast transformation and ribonucleoprotein (RNP) delivery, which enables precise genetic modifications without integrating foreign DNA into the plant genome. This approach leverages the CRISPR/Cas9 system in a transient manner, where preassembled Cas9 protein and guide RNA complexes are introduced into plant cells that have had their walls enzymatically removed [43] [44].

The fundamental advantage of this DNA-free editing strategy lies in its ability to produce genetically edited plants that are indistinguishable from those developed through conventional breeding, potentially streamlining regulatory approval processes [44]. For the Solanum genus, which includes economically critical crops like tomato, potato, and eggplant, this technology offers unprecedented opportunities for rapid trait improvement while mitigating the challenges of Agrobacterium-mediated transformation, particularly the formation of chimeric plants and the obligatory removal of exogenous DNA through extensive backcrossing [43]. As plant biotechnology increasingly focuses on precise genetic improvements, protoplast transformation and RNP delivery have emerged as essential components in the toolkit of modern plant breeders and researchers.

Key Advantages of RNP-Mediated Editing

The utilization of preassembled CRISPR/Cas9 ribonucleoprotein complexes for genome editing offers several distinct advantages over DNA-based delivery methods, particularly in the context of plant biotechnology and crop improvement.

• Elimination of Transgene Integration: RNP-mediated editing functions without inserting foreign DNA into the plant genome, resulting in plants that are not classified as transgenic [43]. This addresses a significant regulatory hurdle in many countries with strict GMO policies [1].
• Reduced Off-Target Effects: The transient presence of RNPs within plant cells minimizes prolonged Cas9 activity, thereby decreasing the probability of unintended mutations [43] [44]. The rapid degradation of RNPs following transfection provides a built-in mechanism for limiting nuclease activity.
• No Codon Optimization Required: Using purified Cas9 protein eliminates the need for plant-specific codon optimization that is typically necessary for DNA-based CRISPR systems [43]. This simplifies vector design and increases the portability of the system across different plant species.
• Simplified Experimental Workflow: The RNP approach bypasses the need for complex binary vector construction to insert T-DNA for expressing Cas9 and gRNA [43]. This reduces both the time and resources required for experimental setup.
• Lower Mosaicism: Direct delivery of functional RNPs leads to more synchronous editing events in transfected cells, resulting in lower rates of chimerism in regenerated plants compared to methods requiring in vivo transcription and translation [43].

Protoplast Isolation and Transformation Workflow

The process of protoplast transformation for transgene-free editing involves multiple critical stages, each requiring optimization for specific plant species. The workflow can be divided into three primary phases: protoplast isolation, transfection with CRISPR/RNPs, and regeneration of whole plants.

Figure 1: Experimental workflow for protoplast transformation and RNP delivery, showing the three main stages from protoplast isolation to regeneration of transgene-free edited plants.

Protoplast Isolation

Successful protoplast isolation requires careful selection of plant material and optimization of enzymatic digestion conditions. Young leaves or hypocotyls from in vitro-grown plants are typically preferred as their cell walls are thinner and more susceptible to enzymatic digestion [43]. For cannabis protoplast isolation, 15-day-old leaves have been shown to yield optimal results, producing approximately 2.2 × 10^6 protoplasts per gram of fresh weight with 78.8% viability [45].

The enzyme solution composition is critical for efficient cell wall digestion without compromising protoplast viability. A typical enzyme mixture includes cellulase (1.5-2% w/v) to hydrolyze cellulose, macerozyme (0.6% w/v) to break down pectin, and pectinase (2% w/v) for comprehensive cell wall digestion [43] [46]. The inclusion of osmotic stabilizers such as mannitol (0.4 M) is essential to prevent osmotic shock to the fragile protoplasts [43] [1]. Calcium chloride is often added to stabilize the plasma membrane and facilitate subsequent fusion processes [43].

RNP Complex Assembly and Transfection

The delivery of CRISPR components as preassembled ribonucleoprotein complexes represents the most advanced approach for DNA-free genome editing. RNP complexes are typically assembled by combining purified Cas9 protein with synthetic single-guide RNA (sgRNA) or crRNA:tracrRNA duplexes. For carrot protoplast transformation, researchers assembled RNPs by mixing 200 pmol of sgRNA with 20 μg of Cas9-GFP protein in 1X PBS buffer, followed by incubation at room temperature for 10 minutes to allow complex formation [47].

Polyethylene glycol (PEG)-mediated transfection is the most commonly used method for introducing RNPs into protoplasts. This approach involves gently mixing the protoplast-RNP mixture with freshly prepared 40% PEG solution, incubating for 15 minutes at room temperature, and then carefully diluting with W5 solution to stop the transfection process [47]. Optimization of PEG concentration and incubation time is crucial for balancing transfection efficiency with protoplast viability. For coconut protoplasts, researchers achieved 48.3% transformation efficiency using 40% PEG-4000 with 0.4 M CaCl₂ and a 30-minute incubation period [46].

Species-Specific Applications and Efficiencies

Protoplast transformation and RNP delivery have been successfully implemented across a diverse range of plant species, with varying efficiencies and regeneration capabilities. The table below summarizes key experimental parameters and outcomes from recent studies.

Table 1: Comparison of Protoplast Transformation and RNP Delivery Efficiency Across Plant Species

Plant Species	Protoplast Yield	Transfection Efficiency	Editing Efficiency	Key Factors for Success
Brassica carinata [1]	Not specified	40% (GFP marker)	Not specified	Five-stage regeneration protocol with specific PGR ratios for each stage
Carrot [47]	Not specified	Not specified	17.28% (sgRNA1), 6.45% (sgRNA2)	RNP delivery with two different sgRNAs targeting invertase gene
Coconut [46]	6.1 × 10^6/g FW	48.3%	4.02%	Optimized enzyme solution (3% cellulase, 1.5% macerozyme, 2% pectinase)
Oil Palm [48]	Not specified	Not specified	24.4-29.1% (plantlets)	Biolistic delivery to embryogenic calli, reduced regeneration time by 4-fold
Cannabis [45]	2.2 × 10^6/g FW	28%	Not specified	Young donor tissue (15-day-old leaves), optimized enzyme solution

Table 2: Media Composition for Different Stages of Protoplast Regeneration in Brassica carinata [1]

Medium Stage	Auxin Concentration	Cytokinin Concentration	Purpose	Key Components
MI	High (NAA + 2,4-D)	Low	Cell wall formation	High auxin concentration
MII	Lower relative to cytokinin	Higher relative to auxin	Active cell division	Balanced auxin:cytokinin ratio
MIII	Low	High (high ratio)	Callus growth and shoot induction	High cytokinin-to-auxin ratio
MIV	Very low	Very high (higher ratio)	Shoot regeneration	Very high cytokinin-to-auxin ratio
MV	Not specified	Low (BAP + GA₃)	Shoot elongation	Low cytokinin with gibberellin

The Scientist's Toolkit: Essential Reagents and Materials

Successful implementation of protoplast transformation and RNP delivery requires specific reagents and materials optimized for each step of the process. The following table details key research reagent solutions essential for these applications.

Table 3: Essential Research Reagent Solutions for Protoplast Transformation and RNP Delivery

Reagent/Material	Function	Example Concentrations	Notes
Cellulase Onozuka R10 [1] [45]	Digest cellulose in plant cell walls	1.5-3% (w/v)	Concentration depends on species and tissue type
Macerozyme R10 [1]	Digest pectin in plant cell walls	0.6-1.5% (w/v)	Often used in combination with cellulase
Mannitol [1] [47]	Osmotic stabilizer	0.4 M	Maintains osmotic balance to prevent protoplast rupture
Polyethylene Glycol (PEG) [47] [46]	Membrane fusion agent for transfection	40% (w/v)	PEG-4000 commonly used; concentration affects efficiency
Cas9 Protein [47] [49]	CRISPR nuclease for targeted DNA cleavage	10 μg/μL	Commercial sources available (e.g., IDT)
Synthetic sgRNA [47] [49]	Guides Cas9 to specific genomic loci	100 μM stock	Chemically synthesized or in vitro transcribed
MES Buffer [1]	pH stabilization	10 mM, pH 5.7	Maintains optimal pH for enzyme activity
Calcium Chloride (CaCl₂) [43] [46]	Membrane stabilization, fusion facilitation	1-125 mM	Concentration varies by application

Critical Factors for Successful Regeneration

The regeneration of whole plants from transfected protoplasts represents the most challenging technical bottleneck in the transgene-free editing pipeline. Successful regeneration requires meticulous optimization of culture conditions and plant growth regulators throughout distinct developmental stages.

Culture Conditions and Media Optimization

Appropriate osmotic pressure maintenance during early culture stages is crucial for protoplast viability and cell wall reformation. For Brassica carinata, researchers developed a highly efficient five-stage regeneration protocol with specific media formulations for each developmental phase [1]. The initial medium (MI) requires high concentrations of auxins (NAA and 2,4-D) to promote cell wall formation, while subsequent media require precisely balanced ratios of cytokinins to auxins for active cell division (MII), callus growth and shoot induction (MIII), shoot regeneration (MIV), and finally shoot elongation (MV) [1].

The duration of culture on different media significantly impacts regeneration success. For cannabis protoplasts, embedding in agarose coupled with a nutrient-rich culture medium containing appropriate plant growth regulators was critical for initiating cell wall re-synthesis (achieving 56.1% efficiency in viable cells), followed by cell division (15.8% plating efficiency) [45]. These findings highlight the species-specific nature of regeneration protocols and the importance of systematic optimization.

Alternative Delivery Methods

While PEG-mediated transfection of protoplasts is widely used, alternative delivery methods have emerged to address limitations in efficiency and regeneration challenges. Biolistic delivery of RNPs directly into meristematic tissues has been successfully employed in species where protoplast regeneration remains challenging. In oil palm, biolistic transformation of embryogenic calli with CRISPR-RNPs achieved mutation frequencies of 24.4-29.1% in rooted plantlets while reducing regeneration time fourfold compared to conventional transgenic methods [48].

Similarly, an in planta particle bombardment-ribonucleoprotein (iPB-RNP) approach was developed for melon, where CRISPR/Cas9 RNPs coated onto gold particles were delivered directly into shoot apical meristem tissue [49]. This method successfully bypassed cell culture requirements altogether, overcoming limitations related to genotype dependence and somaclonal variation. The resulting cmaco1 mutants exhibited significantly extended shelf life due to reduced ethylene production during fruit ripening [49].

Another innovative approach involves grafting wild-type shoots to transgenic donor rootstocks that produce mobile Cas9 and gRNA transcripts. This system has demonstrated heritable gene editing in wild-type Arabidopsis thaliana and Brassica rapa without the need for transgene elimination, culture recovery, and selection [50]. The grafting technique represents a particularly promising avenue for perennial plants and tree species where regeneration from protoplasts remains challenging.

Protoplast transformation combined with RNP delivery represents a powerful platform for transgene-free genome editing in plants. While significant challenges remain, particularly in the regeneration of whole plants from transfected protoplasts for recalcitrant species, continued optimization of isolation, transfection, and regeneration protocols has expanded the application of this technology across diverse crop species. The development of alternative delivery methods, including biolistic RNP delivery and grafting-based systems, provides complementary approaches that may overcome current limitations. As these technologies mature, they promise to accelerate crop improvement efforts by enabling precise genetic modifications without transgene integration, potentially streamlining regulatory processes and public acceptance of genome-edited crops.

The functional characterization of genes in plants, particularly in the context of CRISPR-Cas genome editing, requires robust and efficient validation systems. Hairy root transformation, mediated by Agrobacterium rhizogenes, has emerged as a powerful rapid in planta system for evaluating somatic editing events, bypassing the need for time-consuming stable plant transformation. This system allows for the generation of transgenic roots at the site of infection within weeks, providing ample tissue for molecular analysis of gene editing efficiency. When combined with CRISPR/Cas technology, hairy roots become an indispensable tool for plant functional genomics, enabling rapid assessment of gene function, especially in species that are recalcitrant to conventional transformation or have long life cycles [51] [52]. This protocol outlines standardized methods for utilizing hairy root transformation as a rapid system to evaluate CRISPR/Cas editing efficiency across a diverse range of plant species.

Key Applications in Plant Research

The hairy root transformation system provides a versatile platform for multiple research applications in plant biotechnology. Its utility extends beyond mere transformation to encompass critical functional genomics and metabolite production roles.

• Rapid Validation of CRISPR/Cas Systems: Hairy roots serve as an efficient primary screen for evaluating the activity and efficiency of CRISPR/Cas constructs before embarking on full plant transformation. This application has been successfully demonstrated in cotton, citrus, and medicinal plants, significantly reducing the time and resources required for optimization [53] [52].
• Functional Genomics and Gene Characterization: This system enables rapid investigation of gene function in planta. For example, in licorice (Glycyrrhiza uralensis), hairy root transformation was used to validate the role of GuUGT1 in liquiritin accumulation through both overexpression and CRISPR-mediated knockout studies [54].
• Secondary Metabolite Production: Transgenic hairy roots can be harnessed as sustainable biofactories for valuable secondary metabolites. This is particularly valuable for medicinal plants like cannabis, where hairy root cultures can produce cannabinoids and other therapeutic compounds without cultivating the whole plant [55] [56].
• Transgene-Free Editing: Emerging approaches utilize mobile transcript systems where editing components expressed in hairy roots can move to aerial parts, potentially generating transgene-free edited shoots—a particularly valuable application for woody plants with long breeding cycles [57].

Comparative Efficiency Across Plant Species

Hairy root transformation efficiency varies significantly across plant species, cultivars, and experimental methodologies. The table below summarizes documented transformation efficiencies from recent studies:

Table 1: Hairy Root Transformation Efficiencies Across Plant Species

Plant Species	Agrobacterium Strain	Transformation Efficiency	Key Applications	Citation
Arabidopsis thaliana	C58C1	94% (1-month-old), 75% (6-week-old)	Regeneration studies, cytogenetic analysis	[58]
Cardamine hirsuta	C58C1	93%	Regeneration studies	[58]
Asperuginoides axillaris	C58C1	33%	Conservation of rare species, chromosome analysis	[58]
Cotton	K599	>90%	CRISPR/Cas system validation	[53]
Citrus	K599	Highly efficient (2-8 week protocol)	Genome editing, virus-induced gene silencing	[52]
Cannabis	A4	90% (two-step ex vitro method)	Secondary metabolite production	[56]
Six medicinal plant species across four families	K599	Successful transformation reported	Gene function studies (e.g., GuUGT1)	[54]

Experimental Protocols

Protocol 1: Ex Vitro Two-Step Transformation for High-Throughput Applications

This highly efficient method is ideal for species like cannabis where maintaining mother plants is feasible and high-throughput transformation is desired [56].

• Plant Material Preparation: Maintain healthy mother plants (e.g., 12-week-old cannabis) under optimal greenhouse conditions with regular irrigation and nutrition.
• Agrobacterium Preparation:
  • Use A. rhizogenes strain A4 harboring the binary vector with desired CRISPR/Cas constructs and visible markers (e.g., RUBY).
  • Prepare a dense bacterial lawn by plating 300 μL of bacterial glycerol stock on YEP solid plates containing appropriate antibiotics (e.g., 50 mg/L kanamycin).
  • Incubate plates overnight at 28°C until a thick bacterial lawn forms.
• Plant Transformation:
  • Gently wound nodal sections of the mother plant using a sterile needle.
  • Apply the dense bacterial lawn directly to the wounded sites using a sterile inoculation tool.
  • Maintain inoculated plants in a controlled environment (26°C day/22°C night, 16h light/8h dark, 80% humidity).
  • Continue regular irrigation, periodically misting the wounded area with sterilized distilled water to maintain humidity.
• Hairy Root Monitoring and Isolation:
  • Initial hairy roots typically emerge within 2-4 weeks post-inoculation.
  • Monitor for marker gene expression (e.g., red betalain pigment for RUBY) to identify successfully transformed roots.
  • Excise positive hairy roots when they reach ≥1 cm in length for further analysis.
• Transformation Efficiency Calculation:
  • Record the number of plants with transgenic roots expressing the marker gene.
  • Calculate efficiency as: (Number of plants with transgenic roots / Total number of inoculated plants) × 100.

Protocol 2: Non-Sterile Hypocotyl Transformation for Rapid Validation

This simplified protocol is particularly suitable for rapid validation of CRISPR/Cas systems in cotton and similar species, eliminating the need for sterile conditions [53].

• Seedling Preparation:
  • Germinate seeds directly in potting mix under non-sterile conditions.
  • Grow seedlings until cotyledons are fully expanded.
• Plant Preparation:
  • Decapitate seedlings with a slanted cut, retaining approximately 1 cm of the apical portion of the hypocotyl.
• Agrobacterium Preparation:
  • Use A. rhizogenes strain K599 containing the CRISPR/Cas binary vector.
  • Grow bacterial culture in YEP medium with appropriate antibiotics to OD600 = 0.5-0.6.
• Inoculation:
  • Apply the bacterial suspension directly to the cut surface of the hypocotyl.
  • Maintain plants under controlled environmental conditions.
• Hairy Root Development:
  • Hairy roots typically emerge within 8 days post-inoculation.
  • Transformation efficiency typically exceeds 90% across cotton varieties [53].
• CRISPR/Cas Validation:
  • Harvest transgenic roots for molecular analysis (PCR, sequencing) to assess editing efficiency at target loci.

Protocol 3: Injection-Based Transformation for Brassicaceae Species

This method has been successfully optimized for various Brassicaceae species, including Arabidopsis thaliana, Cardamine hirsuta, and the rare species Asperuginoides axillaris [58].

• Plant Material:
  • Use 4-6 week-old plants with well-developed inflorescence stems.
• Agrobacterium Preparation:
  • Use A. rhizogenes strain C58C1 containing the Ri plasmid and CRISPR/Cas binary vector.
  • Suspend bacteria in infiltration medium to an appropriate density.
• Transformation:
  • Using a sterile syringe, inject the bacterial suspension into the base of the inflorescence primary stem.
  • Avoid excessive damage that may kill the plant.
• Hairy Root Development:
  • Hairy roots emerge at injection sites within 2-4 weeks.
  • Excise emerging roots and culture on solid MS medium with B5 vitamins.
• Plant Regeneration (Optional):
  • Transfer hairy roots to regeneration medium (RGM) containing auxin (8 mg/L NAA) and cytokinin (5 mg/L BAP) to induce shoot formation.
  • Transfer developing shoots to shoot elongation medium (SEM) for 2-3 weeks.
  • Root induced shoots on root induction medium (RIM) before transferring to soil.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Reagents for Hairy Root Transformation and CRISPR/Cas Validation

Reagent/Solution	Function/Purpose	Example Usage/Concentration
Agrobacterium rhizogenes Strains	Delivery of T-DNA containing CRISPR/Cas components	K599 (legumes, cotton, citrus), A4 (cannabis), C58C1 (Brassicaceae) [51] [54] [53]
Binary Vectors	Carries expression cassettes for CRISPR/Cas components	Contains Cas9/gRNA expression units; often with plant-specific promoters [51]
Visible Markers (RUBY)	Visual identification of transformed tissues without destructive sampling	Produces red betalain pigment; enables non-invasive screening [54] [56]
Fluorescent Markers (eGFP, GFP)	Visual identification of transformed tissues	Allows fluorescence-based screening of transgenic roots [53] [52]
Selectable Markers	Selection of transformed tissues	Antibiotic resistance genes (e.g., kanamycin) for in vitro selection [51]
MS Medium with B5 Vitamins	Base medium for hairy root culture and regeneration	Supports root growth and shoot regeneration in various species [58]
Hormone Cocktails for Regeneration	Induces shoot formation from hairy roots	NAA (8 mg/L) + BAP (5 mg/L) for Brassicaceae species [58]

Workflow Visualization

Workflow for CRISPR/Cas Validation Using Hairy Root System

Critical Factors for Success

Several technical considerations significantly impact the efficiency and success of hairy root transformation for somatic editing evaluation:

• Strain Selection: The choice of A. rhizogenes strain significantly influences transformation efficiency. Strain K599 shows high efficiency in legumes, cotton, and citrus; A4 works well in cannabis; while C58C1 is effective in Brassicaceae species [51] [56] [52].
• Marker Systems: Visible markers like RUBY dramatically improve screening efficiency by enabling non-invasive identification of transformed roots without specialized equipment [54] [56].
• Plant Growth Conditions: Optimal physiological status of the plant material is crucial. Younger plants (e.g., 1-month-old Arabidopsis) generally show higher transformation efficiency than older plants (e.g., 6-week-old) [58].
• Vector Design: The choice of promoters driving Cas and gRNA expression affects editing efficiency. Constitutive promoters like 35S are commonly used, but tissue-specific or inducible promoters may offer advantages for certain applications [51].

Hairy root transformation provides researchers with a rapid, efficient, and versatile system for validating CRISPR/Cas editing events in somatic tissues before committing to lengthy full-plant transformation protocols. The methodologies outlined here—ranging from high-throughput ex vitro approaches to simplified non-sterile techniques—offer flexible solutions for diverse research needs and plant species. By enabling rapid functional validation of gene edits and accelerating the characterization of gene function, this system significantly advances plant genomics research and facilitates the development of improved crop varieties with enhanced traits. As CRISPR technologies continue to evolve, hairy root transformation will remain an essential component of the plant biotechnologist's toolkit for somatic editing evaluation.

Application Notes

The integration of CRISPR-based genome editing with plant tissue culture is a cornerstone of modern crop improvement, enabling the direct enhancement of traits such as disease resistance, herbicide tolerance, and resilience to abiotic stresses. The following applications demonstrate the scope and efficacy of this approach.

Enhancing Disease Resistance via Transcriptional Activation

CRISPR activation (CRISPRa) represents a gain-of-function strategy to boost plant immunity without altering the underlying DNA sequence. This system uses a deactivated Cas9 (dCas9) fused to transcriptional activators to upregulate the expression of endogenous defense genes [59].

• Key Application: CRISPRa has been successfully deployed to enhance resistance to bacterial pathogens in staple crops. In tomato, upregulation of the SlPR-1 (PATHOGENESIS-RELATED GENE 1) gene provided enhanced defense against Clavibacter michiganensis. Similarly, epigenetic reprogramming to upregulate the SlWRKY29 gene improved somatic embryogenesis and defense maturation [59].
• Advantage: This method activates genes in their native genomic context, minimizing positional effects and preserving genome integrity compared to transgene-based overexpression [59].

Conferring Herbicide Tolerance through Metabolic Gene Identification and Editing

Genomic studies of weedy relatives of crops can identify key genes responsible for herbicide detoxification, which can then be targeted for editing in cultivated varieties.

• Key Application: Population genomics of weedy oat (Avena fatua) identified a highly differentiated haplotype on chromosome 4D containing an expanded cluster of Glutathione S-transferase (GST) genes. Multi-omics profiling and functional validation demonstrated that one expanded GST gene significantly contributes to strong herbicide resistance [60]. Editing cultivated oat to mimic this haplotype could introduce a robust tolerance mechanism.
• Supporting Evidence: Proteomic analysis of herbicide-resistant winter wild oat (Avena ludoviciana) confirmed the upregulation of detoxification proteins, particularly Cytochrome P450 (CYP450), which plays a direct role in herbicide metabolism [61].

Improving Abiotic Stress Resilience via uORF Engineering

Editing upstream Open Reading Frames (uORFs) is an emerging strategy to fine-tune the expression of genes involved in stress responses without yield penalties.

• Key Application: In Brassica napus (oilseed rape), CRISPR/Cas9 was used to edit uORFs in the BnVTC2 gene, which is involved in ascorbic acid (AsA) biosynthesis. The edited mutants exhibited significantly increased AsA content in leaves, buds, and stems, and demonstrated enhanced tolerance to low temperature, salinity, and drought. Critically, no obvious yield penalty was observed [62].
• Advantage: uORF sequences are often highly conserved across genera, making this a broadly applicable strategy for improving abiotic stress resistance in crops [62].

Table 1: Quantitative Data from CRISPR-Mediated Trait Enhancement Studies

Trait Category	Target Gene/System	Host Plant	Key Quantitative Outcome	Reference
Herbicide Tolerance	GST locus on Chr. 4D	Weedy Oat (A. fatua)	Identification of an expanded GST gene cluster contributing to metabolic resistance.	[60]
Abiotic Stress	BnVTC2 uORF	Brassica napus	Increased Ascorbic Acid; Tolerance to drought, salinity, low temperature; No yield penalty.	[62]
Disease Resistance	CRISPRa-dCas9-TV system	Phaseolus vulgaris (hairy roots)	6.97-fold upregulation of the Pv-lectin defense gene.	[59]
Disease Resistance	CRISPRa-driven SlPR-1	Tomato (S. lycopersicum)	Enhanced defense against Clavibacter michiganensis infection.	[59]

Protocols

Protocol: A Workflow for Validating Herbicide Resistance Genes in Cereals

This protocol outlines steps from genomic identification to functional validation of non-target-site herbicide resistance genes, applicable to cereals like oat.

1. Genomic Identification:

• Population Sequencing: Perform whole-genome sequencing of a large panel (e.g., n=768) of resistant (weedy) and susceptible (cultivated) accessions to construct a genomic variation map [60].
• Variant Analysis: Conduct population genomics analyses to identify genomic loci that are highly divergent and associated with the resistant phenotype. Focus on regions enriched with genes related to biotic/abiotic stress and detoxification (e.g., GSTs, CYP450s) [60].

2. Functional Validation via Transgenic Assay:

• Vector Construction: Clone the candidate resistance gene (e.g., the identified GST) into an appropriate overexpression vector under a strong constitutive promoter like ZmUbi1 [60] [63].
• Plant Transformation: Introduce the construct into a susceptible oat cultivar using established Agrobacterium-mediated transformation or protoplast transformation [60].
• Phenotypic Screening: Subject T0 and T1 transgenic lines to herbicide application. A significant increase in the herbicide dose required to cause 50% growth reduction (ED₅₀) in transgenic plants compared to controls confirms the gene's role in resistance [60] [61].

Protocol: Developing an Efficient CRISPR-Cas9 System for Recalcitrant Species

Many crops, especially perennials and woody trees, are recalcitrant to regeneration in tissue culture. This protocol details the optimization of a CRISPR system using endogenous promoters to overcome this bottleneck.

1. Protoplast Preparation and Transformation Optimization:

• Tissue Source: Use fresh, healthy leaves or embryogenic calli. Optimize enzyme solution (cellulase, macerozyme) concentration and digestion time (typically 4-16 hours) to achieve high yield and viability of protoplasts [63].
• Transfection: Use PEG-mediated transfection for DNA delivery. Critical parameters to optimize include PEG concentration, plasmid DNA amount, and incubation time. Aim for a transient transformation efficiency of at least 40% as a baseline for editing system evaluation [63].

2. Identification and Testing of Endogenous Promoters:

• Screening: Analyze integrated whole-genome and transcriptome sequencing data from the target species to identify highly and constitutively expressed mRNA transcripts [63].
• Cloning and Testing: Clone the upstream regulatory sequences (candidate promoters) of these transcripts into a vector driving a reporter gene (e.g., GFP). Test their expression strength via transient expression in protoplasts compared to standard promoters like CaMV 35S or ZmUbi1 [63].

3. Configuration of the Editing System:

• Vector Assembly: Construct a Single Transcription Unit (STU-Cas9) system where the candidate endogenous promoter drives the expression of both the Cas9 nuclease and the sgRNA(s) on a single cassette. Studies in larch showed that an STU system (LarPE004::STU-Cas9) was more efficient than a Two Transcription Unit (TTU) system and significantly outperformed systems using heterologous promoters [63].
• Multiplexing: For multiple gene knockouts, construct the STU vector with multiple sgRNA expression cassettes in tandem to enable efficient editing of several targets simultaneously [63].

Table 2: Research Reagent Solutions for CRISPR-Tissue Culture Workflows

Reagent/Material	Function in Workflow	Example & Notes
Endogenous Promoters	Drives high, constitutive expression of Cas9/sgRNA in host plant.	e.g., LarPE004 promoter from larch; superior to CaMV 35S in monocots and recalcitrant species [63].
STU-Cas9 Vector System	Single Transcription Unit for coordinated expression of Cas9 and sgRNA.	Improves editing efficiency compared to TTU systems; ideal for multiplex editing [63].
PAM-Relaxed Cas Variants	Expands the range of targetable genomic sites.	SpRY mutant protein enables editing across various PAM sites, increasing targeting flexibility [63].
Protoplast Transformation System	Enables rapid testing of editing efficiency without stable transformation.	PEG-mediated transfection of optimized protoplasts is a key high-throughput screening step [63].
Temporary Immersion System (TIS)	Automated bioreactor for scaling up regenerated plantlets.	Systems like BioCoupler improve lab efficiency and biomass production during regeneration [64].

Pathway and Workflow Visualizations

CRISPR Activation (CRISPRa) for Disease Resistance

Integrated Workflow: From Gene Editing to Regenerated Plant

The integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with advanced plant tissue culture techniques is revolutionizing plant biotechnology. This synergy enables precise genetic modifications and the regeneration of improved crop varieties, directly supporting global food security efforts. This article presents detailed application notes and protocols for successful CRISPR editing in four key species: rice, soybean, poplar, and tomato, providing researchers with practical frameworks for genetic improvement workflows.

Comparative Analysis of CRISPR Editing Across Species

Table 1: Summary of CRISPR-Cas Editing Applications in Rice, Soybean, Poplar, and Tomato

Species	Target Gene(s)	Editing Purpose	Delivery Method	Tissue Culture/Regeneration System	Key Editing Outcome	Efficiency/Key Metric
Rice (Tropical Japonica)	Phytoene Desaturase (PDS), Young Seedling Albino (YSA) [65]	Gene knockout validation with visible phenotypic markers (albino phenotype) [65]	Agrobacterium-mediated transformation & particle bombardment [65]	Callus induction from mature seeds [65]	Successful knockout confirmed by albino phenotype and Sanger sequencing (indels) [65]	33% regeneration efficiency; 60% of regenerated PDS plants showed albino phenotype [65]
Soybean	Various for shoot architecture (e.g., node number, internode length) [66]	Optimize plant architecture for enhanced yield potential [66]	Agrobacterium-mediated transformation; RNP-based systems (promising) [67]	Stable transformation reliant on Agrobacterium; Hairy root assays for gRNA validation [67]	A major challenge is overcoming low transformation efficiency and editing polyploid genomes [67]	Efficiency is a key bottleneck; improved by novel delivery (e.g., viral vectors, RNP) [67] [66]
Poplar	Target genes for fiber production [68]	Increase fiber production capacity [68]	Multiplex CRISPR approach [68]	Information not specified in search results	Successful application of multiplex editing [68]	Information not specified in search results
Tomato	Genome-wide multi-targeted libraries (15,804 sgRNAs) [68]	Overcome functional redundancy in gene families; improve fruit and pathogen traits [68]	Agrobacterium-mediated transformation; Protoplast transfection (RNP/DNA) [68] [69]	Callus-based regeneration; Protoplast regeneration (for transgene-free editing) [68] [69]	Mutants identified for fruit development, flavor, nutrient uptake, and pathogen response [68]	~1300 independent lines generated; mutation detection rate was a focus [68]

Application Notes and Protocols

Tomato: Genome-Wide Multi-Targeted CRISPR Libraries

1. Experimental Objectives and Design Tomato faces significant challenges from functional redundancy within large gene families, which can mask the phenotypic effects of single-gene mutations. This protocol outlines the development of a genome-wide, multi-targeted CRISPR library designed to overcome this redundancy. The library uses single guide RNAs (sgRNAs) that target conserved sequences across multiple members of a gene family, enabling simultaneous editing of several genes to reveal their combined function [68].

2. Tissue Culture and Transformation Workflow

• Explants: Use cotyledons or hypocotyls from sterile tomato seedlings [70].
• Agrobacterium Co-cultivation: Immerse explants in an Agrobacterium tumefaciens suspension carrying the multi-sgRNA library construct for 20-30 minutes.
• Callus Induction: Transfer explants to solid selection medium containing antibiotics (e.g., kanamycin) and hormones (e.g., auxins and cytokinins) to induce callus formation.
• Shoot Regeneration: Subculture developing callus onto shoot induction medium.
• Rooting and Acclimatization: Induce roots on regenerated shoots and acclimatize plantlets to greenhouse conditions [68] [70].

3. Key Findings and Outcomes This approach successfully generated approximately 1,300 independent CRISPR lines. Phenotypic screening identified over 100 independent mutant lines with distinct characteristics related to fruit development, flavor, nutrient uptake, and pathogen response [68]. The study also developed CRISPR-GuideMap, a double-barcode tagging system, to track sgRNAs in generated plants efficiently [68].

Rice: Optimized Transformation for Tropical Japonica Cultivar

1. Experimental Objectives and Design The objective was to overcome the recalcitrance of tropical japonica rice varieties to transformation and regeneration, using the high-yielding cultivar 'Presidio' as a model. The protocol was optimized to achieve efficient CRISPR/Cas9 gene editing by targeting the Phytoene Desaturase (PDS) and Young Seedling Albino (YSA) genes, which produce visible albino phenotypes for easy knockout validation [65].

2. Tissue Culture and Transformation Workflow

• Callus Induction: Induce embryogenic calli from mature seeds on N6-based medium [65].
• Transformation: Use Agrobacterium-mediated transformation or particle bombardment to deliver CRISPR/Cas9 constructs.
• Selection and Regeneration: Transfer calli to selection medium containing hygromycin. Regenerate putative transgenic plants on regeneration medium [65].
• Molecular Analysis: Extract genomic DNA from regenerated plant leaves. Use PCR and Sanger sequencing to detect mutations at the target sites [65].

3. Key Findings and Outcomes The optimized protocol resulted in a 33% regeneration efficiency. For the PDS target, 60% of regenerated plants displayed a clear albino phenotype, confirming successful gene knockout. Sequencing revealed various insertions, deletions, and substitutions at the target sites, validating the protocol's effectiveness for a previously recalcitrant cultivar [65].

Soybean: Editing for Optimized Shoot Architecture

1. Experimental Objectives and Design Soybean improvement aims to optimize shoot architecture traits like node number, internode length, and branching to enhance yield. This protocol involves using CRISPR/Cas systems to edit key genes governing these traits. A significant challenge is soybean's polyploid genome, which requires efficient editing of multiple homologous genes [67] [66].

2. Tissue Culture and Transformation Workflow

• Explants: Use immature cotyledons or half-seeds as explants.
• Agrobacterium Co-cultivation: Infect explants with A. tumefaciens carrying the CRISPR construct.
• Selection and Regeneration: Culture explants on selection medium to generate transgenic events. Regenerate whole plants via organogenesis [67].
• Alternative DNA-Free Approach (Protoplast System):
  • Protoplast Isolation: Isolate protoplasts from soybean cell suspensions or young leaves using cellulase and pectinase.
  • Transfection: Introduce pre-assembled CRISPR-Cas9 Ribonucleoprotein (RNP) complexes via polyethylene glycol (PEG)-mediated transfection.
  • Regeneration: Culture transfected protoplasts to regenerate whole plants, a step that remains challenging but is critical for producing transgene-free edited plants [67].

3. Key Findings and Outcomes CRISPR/Cas9 has been successfully used to target genes controlling plant architecture in soybean. However, low transformation and regeneration efficiency remain major bottlenecks. Emerging delivery systems, such as virus-induced genome editing (VIGE) and RNP-based approaches, show promise for overcoming these challenges and producing transgene-free edited plants [66].

Poplar: Multiplex CRISPR for Trait Enhancement

1. Experimental Objectives and Design In poplar, a perennial tree species, the objective was to apply a multiplex CRISPR strategy to simultaneously edit multiple genes to improve complex traits, such as fiber production for the wood and paper industries [68].

2. Tissue Culture and Transformation Workflow

• Explants: Use leaf discs or hypocotyl segments from sterile seedlings.
• Agrobacterium Co-cultivation: Transform explants with A. tumefaciens carrying a multiplex CRISPR construct containing several sgRNA expression cassettes.
• Regeneration: Regenerate whole plants through organogenesis on medium containing auxins and cytokinins [68].

3. Key Findings and Outcomes The multiplex CRISPR approach in poplar demonstrated the potential for simultaneous editing of multiple genomic loci in one generation, leading to improved traits like increased fiber production. This showcases the power of CRISPR for rapid improvement of long-lifecycle perennial species [68].

Detailed Experimental Protocols

Agrobacterium-Mediated Transformation of Tomato

This is a generalized protocol for generating CRISPR-edited tomato plants via Agrobacterium-mediated transformation, a widely used and efficient method [70].

Diagram Title: Tomato Agrobacterium Transformation Workflow

Protocol Steps:

• Seed Sterilization and Germination:
  • Surface-sterilize tomato seeds with 70% ethanol for 1 minute, then 2% sodium hypochlorite for 15 minutes, followed by 3-5 rinses with sterile distilled water.
  • Sow seeds on solid MS basal medium and germinate under a 16/8 h light/dark photoperiod at 25°C [70].

• Explant Preparation:

  • After 10-14 days, use sterile seedlings. Excise cotyledons and hypocotyls into 5-8 mm segments for use as explants [70].

• Agrobacterium Preparation and Co-cultivation:

  • Grow Agrobacterium tumefaciens strain GV3101 harboring the CRISPR/Cas9 binary vector in YEP medium with appropriate antibiotics to an OD₆₀₀ of 0.5-0.8.
  • Resuspend the bacterial pellet in liquid MS or co-cultivation medium.
  • Immerse explants in the Agrobacterium suspension for 20-30 minutes with gentle shaking.
  • Blot-dry explants and transfer them to co-cultivation medium for 2-3 days in the dark at 25°C [70].

• Selection and Callus Induction:

  • After co-cultivation, wash explants 2-3 times with sterile distilled water containing carbenicillin (500 mg/L) to eliminate Agrobacterium.
  • Transfer explants to selection medium (e.g., containing kanamycin) supplemented with carbenicillin and hormones for callus induction. Subculture every two weeks [70].

• Shoot Regeneration and Rooting:

  • Transfer developed callus to shoot regeneration medium. Regenerated shoots (2-3 cm long) are transferred to rooting medium.
  • Once a healthy root system develops, carefully acclimatize plantlets to greenhouse conditions [70].

• Molecular Analysis:

  • Extract genomic DNA from regenerated plant leaves using a CTAB-based method.
  • Perform PCR to amplify the target region and sequence the amplicons to identify mutations [71] [70].

DNA-Free Editing in Tomato via Protoplast Transfection

This protocol describes a transgene-free genome editing method using direct delivery of CRISPR-Cas9 Ribonucleoprotein (RNP) complexes into tomato protoplasts, bypassing the need for Agrobacterium and avoiding T-DNA integration [69].

Diagram Title: DNA-Free Tomato Editing via Protoplasts

Protocol Steps:

• Protoplast Isolation:
  • Select young leaves or hypocotyls from sterile in vitro tomato plants.
  • Thinly slice tissue and immerse in an enzyme solution (e.g., 1.5% cellulase, 0.5% macerozyme, 0.4 M mannitol, 20 mM KCl, 20 mM MES pH 5.7, 10 mM CaCl₂).
  • Digest for 6-16 hours in the dark with gentle shaking (30-40 rpm).
  • Filter the mixture through a 50-100 μm mesh and centrifuge to purify protoplasts. Resuspend in W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES pH 5.7). Assess viability using fluorescein diacetate (FDA) staining [69].

• RNP Complex Assembly and Transfection:

  • Assemble RNP complexes in vitro by incubating purified Cas9 protein with synthetic sgRNA targeting the gene of interest.
  • Mix ~2x10⁵ protoplasts with the RNP complexes in a transfection solution containing PEG (e.g., 40% PEG4000). Incubate for 15-30 minutes.
  • Stop the reaction by diluting with W5 solution and pellet the protoplasts [69].

• Protoplast Culture and Regeneration (Bottleneck):

  • Culture transfected protoplasts at high density in liquid or alginate-solidified culture medium (e.g., KM or MS based with hormones).
  • Monitor for cell wall regeneration and initial cell divisions. Transfer microcalli to solid callus induction medium.
  • Regenerate shoots and roots via standard tissue culture protocols. This regeneration step is often the most challenging and species-dependent [69].

• Mutation Analysis:

  • To assess editing efficiency early, extract genomic DNA from a portion of transfected protoplasts 2-3 days after transfection.
  • Use targeted amplicon sequencing (the "gold standard" for sensitivity and accuracy) or PCR/restriction fragment length polymorphism (PCR-RFLP) assays to detect and quantify mutations [71] [69].

The Scientist's Toolkit: Essential Reagents and Solutions

Table 2: Key Research Reagent Solutions for CRISPR Plant Tissue Culture

Reagent Category	Specific Examples	Function in Workflow
CRISPR Components	Cas9 protein (for RNP), Cas9-expression vectors, sgRNA scaffolds [69]	The core editing machinery; Cas9 nuclease creates double-strand breaks, guided by sgRNA to specific genomic loci.
Transformation Vectors	Binary Vectors (for Agrobacterium), Geminiviral Replicons (for transient amplification) [66]	Deliver and/or express CRISPR components within plant cells.
Enzymes for Protoplasting	Cellulase, Macerozyme, Pectinase [69]	Digest plant cell walls to create protoplasts for RNP or DNA transfection.
Culture Media Bases	MS (Murashige and Skoog), N6 (for cereals) Medium [65]	Provide essential nutrients, vitamins, and minerals to support plant cell growth and regeneration in tissue culture.
Growth Regulators	Auxins (2,4-D, IAA), Cytokinins (BAP, Zeatin) [70]	Control cell division, callus formation, and organogenesis (shoot and root initiation).
Selection Agents	Antibiotics (Kanamycin, Hygromycin), Herbicides [70]	Select for successfully transformed cells by eliminating non-transformed tissue.
Detection & Analysis Kits	PCR Kits, Restriction Enzymes (for RFLP), T7E1/SURVEYOR Assay Kits, Sanger Sequencing Services [71]	Confirm genetic edits, analyze mutation efficiency, and detect zygosity.

The detailed application notes and protocols for rice, soybean, poplar, and tomato demonstrate the critical role of optimized tissue culture and transformation systems in deploying CRISPR-Cas technology for crop improvement. While challenges remain, particularly in regeneration and editing efficiency for species like soybean, continued advancements in delivery methods, such as RNP-based systems and tissue culture-independent approaches, promise to further enhance the precision and accessibility of plant genome editing. These foundational protocols provide a robust starting point for researchers aiming to contribute to the next generation of improved crop varieties.

Overcoming bottlenecks: Enhancing efficiency and precision in edited plant recovery

Addressing Genotype Dependence and Low Regeneration Efficiency

A significant bottleneck in plant biotechnology, particularly for CRISPR-edited plants, is the reliance on efficient tissue culture and regeneration systems. These processes are often plagued by two interconnected challenges: strong genotype dependence, where protocols work only for a limited number of elite genotypes, and low regeneration efficiency, which drastically slows down the production of edited plants. This application note details three advanced strategies—protoplast regeneration systems, morphogenic regulator-assisted transformation, and tissue culture-free methods—to overcome these barriers. By providing comparative quantitative data, standardized protocols, and underlying signaling pathway insights, this document serves as a practical guide for researchers aiming to achieve high-efficiency regeneration across a wider range of plant species and genotypes.

Comparative Analysis of Advanced Regeneration Strategies

The following table summarizes the core quantitative findings and applications of the three principal strategies discussed in this note.

Table 1: Comparison of Advanced Strategies to Address Regeneration Challenges

Strategy	Reported Efficiency	Key Advantage	Demonstrated Species	Key Regulatory Factors
Optimized Protoplast Regeneration [72]	Regeneration frequency up to 64%; Transfection efficiency 40% (GFP marker)	DNA-free editing potential; High throughput for gRNA validation	Brassica carinata	Specific, stage-dependent NAA, 2,4-D, and Cytokinin ratios
Morphogenic Regulators (GRF-GIF) [73]	Transformation increase up to ~70%; ~2-fold enhancement in regeneration	Genotype-independent transformation; Broader species applicability	Tomato, Wheat, Rice, Lettuce	GRF4-GIF1 chimera (miRNA-resistant)
Tissue Culture-Free (WIND-IPT) [22]	Successful gene-edited shoots generation; Higher regeneration success	Bypasses tissue culture entirely; Faster, less technically demanding	Tobacco, Tomato, Soybean	Combined WIND1 and IPT gene expression

Detailed Experimental Protocols

A Five-Stage Protoplast Regeneration System forBrassica carinata

This protocol enables high-frequency shoot regeneration from protoplasts, providing a foundation for DNA-free genome editing [72].

• 1. Plant Material and Protoplast Isolation

  • Germination: Surface-sterilize seeds and germinate on half-strength MS medium with 10 g L⁻¹ sucrose and 7 g L⁻¹ Bacto agar (pH 5.7). Maintain plants at 25°C (day)/18°C (night) with a 16-hour photoperiod [72].
  • Protoplast Isolation: Harvest fully expanded leaves from 3-4 week-old seedlings. Finely slice leaves and incubate in plasmolysis solution (0.4 M mannitol, pH 5.7) for 30 minutes in the dark. Digest tissue in an enzyme solution containing 1.5% cellulase Onozuka R10, 0.6% Macerozyme R10, 0.4 M mannitol, 10 mM MES, 0.1% BSA, 1 mM CaCl₂, and 1 mM β-mercaptoethanol (pH 5.7) for 14-16 hours in the dark with gentle shaking [72].
  • Purification: Filter the digested mixture through a 40-μm nylon mesh. Centrifuge the filtrate at 100 g for 10 minutes and wash the pellet twice with W5 solution. Resuspend the final protoplast pellet in W5 solution and keep on ice for 30 minutes before adjusting the density to 4-6 x 10⁵ cells mL⁻¹ in 0.5 M mannitol [72].

• 2. Protoplast Transfection

  • Mix the protoplast suspension with an equal volume of 2.8% sodium alginate (in 0.4 M mannitol).
  • Pipette 600 μL of the mixture onto calcium-agar plates (0.4 M mannitol, 2.2 g L⁻¹ CaCl₂, 10 g L⁻¹ Phyto agar) to form thin alginate disks.
  • For transfection, incubate protoplasts with CRISPR/Cas9 ribonucleoproteins (RNPs) or plasmid DNA in the presence of PEG. The use of a GFP marker gene can achieve ~40% transfection efficiency [72].

• 3. Multi-Stage Protoplast Regeneration

  • The following five-stage media regime is critical for efficient regeneration. The specific plant growth regulator (PGR) combinations for each stage are detailed in Table 2.
  • Stage I (MI - Cell Wall Formation): Culture transfected protoplasts embedded in alginate disks on MI medium. This stage requires high auxin concentrations (NAA and 2,4-D) to initiate cell wall formation.
  • Stage II (MII - Active Cell Division): Transfer developing microcalli to MII medium. A lower auxin-to-cytokinin ratio is essential to promote active cell division.
  • Stage III (MIII - Callus Growth & Shoot Induction): Move proliferating calli to MIII medium. A high cytokinin-to-auxin ratio is critical for shoot induction.
  • Stage IV (MIV - Shoot Regeneration): Culture shoot-forming calli on MIV medium. An even higher cytokinin-to-auxin ratio is optimal for robust shoot regeneration.
  • Stage V (MV - Shoot Elongation): Individualize developing shoots and transfer to MV medium. Low levels of BAP and GA₃ are sufficient to promote shoot elongation [72].

Table 2: Media Formulation for the Five-Stage Protoplast Regeneration System

Media Stage	Primary Function	Critical PGRs & Concentrations	Key Osmoticum
MI	Cell Wall Formation	High NAA and 2,4-D	0.4 M Mannitol
MII	Active Cell Division	Lower Auxin : Cytokinin ratio	0.4 M Mannitol
MIII	Callus Growth & Shoot Induction	High Cytokinin : Auxin ratio	Sucrose
MIV	Shoot Regeneration	Very High Cytokinin : Auxin ratio	Sucrose
MV	Shoot Elongation	Low BAP and GA₃	Sucrose

Morphogenic Regulator-Mediated Transformation Enhancement

This protocol utilizes the co-expression of GRF4 and GIF1 to boost transformation and regeneration efficiency in recalcitrant tomato genotypes [73].

• 1. Vector Construction

  • Clone the miRNA-resistant version of the SlGRF4 (SlrGRF4) and SlGIF1 genes, either as separate expression cassettes or as a single SlGRF4-GIF1 chimera, into a binary vector suitable for Agrobacterium-mediated transformation.
  • Incorporate the visible reporter gene RUBY into the T-DNA. RUBY produces a red pigment (betaine) that allows for non-destructive, visual tracking of transformation events without specialized equipment [73].

• 2. Plant Transformation and Regeneration

  • Agrobacterium Co-cultivation: Transform explants (e.g., cotyledons, hypocotyls) from your target tomato genotypes using standard Agrobacterium tumefaciens protocols with the constructed vector.
  • Callus Induction: Culture explants on callus-inducing medium (CIM) containing auxins to induce the formation of pluripotent callus.
  • Shoot Regeneration: Transfer the induced callus to shoot-inducing medium (SIM). The presence of SlGRF4-GIF1 significantly enhances the shoot regeneration capacity across genotypes.
  • Selection and Screening: Identify positive transformation events by the visible red pigmentation from the RUBY reporter. Regenerate and root shoots on appropriate media [73].

Signaling Pathways Governing Plant Regeneration

Understanding the molecular networks that control cell fate is key to rationally improving regeneration. The following diagrams, generated using DOT language, illustrate two critical pathways.

The CLE-CLV1/BAM1 Signaling Module in Shoot Regeneration

This pathway acts as a negative regulator of adventitious shoot formation [74].

Diagram 1: CLE signaling negatively regulates shoot regeneration.

The REF1-PORK1-WIND1 Regulatory Loop

This pathway forms a positive feedback loop that enhances regenerative capacity in response to wounding [74].

Diagram 2: The REF1-PORK1-WIND1 loop promotes regeneration.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for Implementing Advanced Regeneration Protocols

Reagent / Material	Function / Application	Specific Example / Note
Cellulase Onozuka R10	Cell wall digestion for protoplast isolation	Used at 1.5% (w/v) in enzyme solution [72]
Macerozyme R10	Pectin digestion for protoplast isolation	Used at 0.6% (w/v) in enzyme solution [72]
Sodium Alginate	Protoplast embedding for culture	2.8% (w/v) solution for forming alginate disks [72]
GRF4-GIF1 Chimera	Morphogenic regulator to enhance regeneration	miRNA-resistant version (rGRF4-GIF1) shows superior performance [73]
RUBY Reporter	Visual, non-destructive marker for transformation	Betalain pigment; requires no substrates or special equipment [73]
WIND1 & IPT Genes	Key genes for tissue-culture-free transformation	Combined expression triggers shoot regeneration from wound sites [22]
PEG (Polyethylene Glycol)	Facilitates transfection in protoplast systems	Used for delivery of CRISPR RNPs or DNA [72] [75]

Concluding Remarks and Implementation Guide

The integration of the detailed protocols, pathway insights, and reagent toolkit provided herein empowers researchers to systematically address the challenges of genotype dependence and low regeneration efficiency.

For practical implementation:

• For high-throughput gRNA validation where plant regeneration is not the immediate goal, the maize protoplast system [75] offers a rapid solution.
• To develop a stable transformation system for a recalcitrant crop, prioritizing the implementation of morphogenic regulators like GRF4-GIF1 [73] is the most impactful strategy.
• For species with extremely robust tissue culture protocols, the optimized protoplast regeneration system [72] provides a direct path to DNA-free editing.
• The emerging tissue culture-free methods using genes like WIND1 and IPT [22] represent the future of plant transformation and should be closely monitored and adopted as they mature for broader species application.

By leveraging these strategies, the scientific community can accelerate the development of CRISPR-edited plants, paving the way for faster crop improvement and functional genomics research.

Within plant tissue culture and CRISPR research, achieving high editing efficiency is paramount for successfully generating mutant plants. The selection of regulatory elements, particularly promoters, and the overall design of the transformation vector are two fundamental factors that directly influence the outcome of gene editing experiments. This Application Note details the strategic selection of promoters and the implementation of optimized vector designs to maximize editing efficiency in plants, providing researchers with actionable protocols and frameworks.

The Critical Role of Promoter Selection

The promoter drives the expression of the CRISPR-Cas machinery and is a primary determinant of editing success. Different promoters offer varying expression levels, temporal control, and tissue specificity.

Comparative Performance of Promoter Types

The table below summarizes key performance characteristics of different promoter types used in plant CRISPR systems.

Table 1: Characteristics of Promoters for CRISPR-Cas Expression in Plants

Promoter Type	Example	Key Features	Reported Performance
Constitutive	CaMV 35S, ZmUbi1	Strong, ubiquitous expression; widely used.	Often outperformed by highly expressed endogenous promoters [63].
Endogenous	LarPE004 (Larch)	Derived from the host species; matched expression machinery.	In larch, LarPE004::STU-Cas9 system was significantly more efficient than 35S- and ZmUbi1-driven systems [63].
Artificial Intelligence (AI)-Generated	OpenCRISPR-1 (Designed for human cells)	AI-designed effectors tailored for optimal function in non-native environments [76].	Exhibits comparable or improved activity and specificity relative to SpCas9 [76].

Protocol: Evaluating Promoter Efficiency in a Protoplast System

This protocol is adapted from a larch study [63] and can be adapted for other plant species to rapidly screen promoter performance.

1. Protoplast Isolation and Transformation

• Isolate protoplasts from the target plant species or a suitable model using enzymatic digestion.
• Prepare plasmid DNA for each promoter-Cas9 construct to be tested, all targeting the same genomic locus.
• Transferd the protoplasts with the constructed vectors using polyethylene glycol (PEG)-mediated transformation. A common control is a vector expressing a fluorescent protein to determine baseline transformation efficiency.

2. Analysis of Editing Efficiency

• Incubate the transformed protoplasts for 24-48 hours to allow for gene expression and editing.
• Extract Genomic DNA from the protoplast population using a commercial plant DNA extraction kit.
• Amplify the Target Region by PCR using primers flanking the Cas9 cut site.
• Assess Editing using a method of choice:
  • T7 Endonuclease I (T7EI) Assay: Digest the heteroduplexed PCR products with T7EI and analyze via gel electrophoresis. The fraction of cleaved products indicates mutation frequency.
  • Restriction Fragment Length Polymorphism (RFLP) Assay: If the edit disrupts a restriction site, digest the PCR products and analyze the fragment pattern.
  • Sanger Sequencing & Decomposition: Sequence the PCR amplicon and use online tools (e.g., ICE Synthego) to quantify the indel percentage.

Diagram: Workflow for evaluating promoter-driven editing efficiency in protoplasts.

Optimized Vector Design Architectures

The arrangement of CRISPR-Cas components within the transformation vector significantly impacts the consistency and level of editing.

Single vs. Multiple Transcription Unit Designs

The two primary vector architectures are the Single Transcription Unit (STU) and the Two Transcription Unit (TTU) systems.

Table 2: Comparison of CRISPR Vector Architectures

Vector Architecture	Description	Advantages	Considerations
Single Transcription Unit (STU)	Cas9 and gRNA(s) are expressed from a single polycistronic transcript.	Simpler construction; ensures coordinated delivery of all components [63].	May require internal ribosome entry sites (IRES) or self-cleaving peptides (e.g., P2A).
Two Transcription Unit (TTU)	Cas9 and gRNA(s) are expressed from independent transcriptional units, each with its own promoter and terminator.	Allows for independent optimization of Cas9 and gRNA expression levels.	More complex vector assembly; larger DNA construct size.

Evidence from larch indicates that the LarPE004::STU-Cas9 system was more efficient for both single and multiple gene editing than the corresponding TTU system [63].

Protocol: Agrobacterium-Mediated Stable Transformation for Fraxinus mandshurica

This protocol outlines the stable transformation of a woody species, Fraxinus mandshurica, using an optimized CRISPR vector [4].

1. Vector Construction and Agrobacterium Preparation

• Clone the selected efficient promoter (e.g., an endogenous candidate) driving the Cas9 nuclease into a binary vector backbone.
• Clone the gRNA expression cassette, under a U6 snRNA promoter, targeting the gene of interest (e.g., FmbHLH1).
• Introduce the final construct into Agrobacterium tumefaciens strain EHA105 via electroporation or freeze-thaw transformation [4].
• Culture a single colony of the transformed Agrobacterium in LB medium with appropriate antibiotics until it reaches the optimal optical density (OD₆₀₀) of 0.6-0.8 [4].

2. Plant Transformation and Selection

• Use sterile plantlets grown from embryos as explant material.
• Immerse the explants in the Agrobacterium suspension for infection. Optimize the duration of infection for your species.
• Co-cultivate the infected explants on solid medium for 2-3 days to allow for T-DNA transfer.
• Transfer the explants to a selection medium containing antibiotics (e.g., kanamycin at 40-50 mg/L for Fraxinus) to inhibit Agrobacterium growth and select for transformed plant cells, and hormones to induce clustered buds [4].
• Subculture developing shoots onto fresh selection medium every 3-4 weeks until stable lines are established.

3. Screening for Homozygous Mutants

• Induce and screen using a clustered bud system to propagate and enrich for edited events [4].
• Extract genomic DNA from regenerated plantlets and perform PCR on the target region.
• Sequence the PCR products to identify heterozygous and homozygous mutations. In the Fraxinus study, this method resulted in 18% of induced clustered buds being gene-edited [4].

Diagram: Workflow for Agrobacterium-mediated stable transformation and mutant screening.

Advanced Systems and Screening Methods

Expanding the Editing Toolbox

Beyond standard Cas9, new systems are increasing the flexibility and scope of genome editing.

• CRISPR-Activation (CRISPRa): This system uses a deactivated Cas9 (dCas9) fused to transcriptional activators (e.g., VP64) to upregulate endogenous genes without altering the DNA sequence. This is a powerful gain-of-function tool for studying gene function and enhancing traits like disease resistance [59].
• AI-Designed Editors: Machine learning models trained on vast microbial genome datasets can now generate novel CRISPR effectors. The editor "OpenCRISPR-1" is one such AI-generated protein that is highly functional in human cells despite being ~400 mutations away from SpCas9, demonstrating a new paradigm for editor development [76].
• Relaxed PAM Specificity: Cas9 variants like SpRY recognize a broader range of PAM sites, enabling editing of previously inaccessible genomic regions. These can be incorporated into optimized vector systems (e.g., LarPE004::STU-SpRY) for improved targeting capabilities [63].

Protocol: Rapid Evaluation of Editing Outcomes via Fluorescent Protein Conversion

This cell-based protocol uses a phenotypic readout to quickly quantify the efficiency of different repair outcomes [77].

1. Generate Reporter Cell Line

• Create a stable cell line expressing enhanced Green Fluorescent Protein (eGFP) via lentiviral transduction.

2. Transfection and Analysis

• Design a CRISPR-Cas9 system to target the eGFP gene. Co-deliver a repair template to convert eGFP to Blue Fluorescent Protein (BFP) via Homology-Directed Repair (HDR).
• Transfect the eGFP-positive cells with the gene editing reagents.
• After a suitable incubation period, analyze the cells using Fluorescence-Activated Cell Sorting (FACS).
• Interpret the results:
  • Non-fluorescent cells: Indicate a successful gene knockout via Non-Homologous End Joining (NHEJ).
  • Blue fluorescent cells (BFP+): Indicate successful HDR-mediated gene conversion.
  • Remaining green fluorescent cells (eGFP+): Indicate no editing occurred.

This assay allows for the high-throughput, scalable assessment of how promoter/vector choices influence the balance between NHEJ and HDR repair pathways [77].

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for CRISPR-Cas9 Plant Genome Editing

Reagent / Material	Function / Application	Examples / Notes
Binary Vectors	T-DNA backbone for Agrobacterium-mediated plant transformation.	pYLCRISPR/Cas9P35S-N [4]; other plant CRISPR binary vectors.
Cas9 Nuclease	Engineered nuclease that creates double-strand breaks in DNA.	Alt-R S.p. Cas9 Nuclease V3 (IDT) [78]; can be codon-optimized for plants.
Guide RNA Components	Targets the Cas9 nuclease to a specific genomic locus.	Alt-R CRISPR-Cas9 crRNA and tracrRNA (IDT) [78]; can be purchased as custom synthetic RNAs.
Plant Tissue Culture Media	Supports the growth and regeneration of plant cells and tissues.	Woody Plant Medium (WPM) [4]; Murashige and Skoog (MS) Medium.
Selection Agents	Selects for successfully transformed plant cells.	Kanamycin [4], Hygromycin; the concentration must be optimized for each species.
Agrobacterium Strain	Mediates the transfer of T-DNA from the vector into the plant genome.	EHA105 [4], GV3101; the choice of strain can impact transformation efficiency.

Strategies for Multiplex Editing and Complex Trait Stacking

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous modification of multiple genetic loci to address polygenic traits and accelerate crop improvement. This Application Note provides a comprehensive technical overview of current strategies, protocols, and analytical methods for implementing multiplex editing in plant systems. We detail vector design considerations, delivery methods, and mutation detection techniques specifically optimized for complex trait stacking, along with experimental workflows that integrate seamlessly with plant tissue culture pipelines. The protocols outlined support researchers in overcoming key challenges in genetic redundancy, trait pyramiding, and de novo domestication efforts.

Multiplex CRISPR editing represents a powerful approach for simultaneous modification of multiple genes, regulatory elements, or chromosomal regions within a single experiment. This capability is particularly valuable for addressing the polygenic nature of many agronomic traits and overcoming genetic redundancy pervasive in plant genomes [79]. Unlike traditional single-gene editing approaches, multiplex systems enable researchers to dissect gene family functions, engineer complex metabolic pathways, and stack multiple beneficial traits in a coordinated manner. The technology has evolved beyond standard gene knockouts to include epigenetic modulation, transcriptional regulation, and chromosomal engineering, making it indispensable for next-generation crop improvement programs focused on climate resilience and sustainability [79].

The fundamental advantage of multiplex editing lies in its ability to generate higher-order combinatorial mutations that would be impractical to create through sequential editing or conventional breeding. For instance, where durable powdery mildew resistance in dicots requires multigene knockouts, a single multiplex transformation can generate both single- and multi-gene knockouts in various combinations, greatly accelerating research progress [79]. In cucumber, simultaneous knockout of three clade V genes (Csmlo1 Csmlo8 Csmlo11) was necessary to achieve full resistance, demonstrating how multiplex approaches can address genetic compensation [79].

Multiplexing Strategies and Vector Systems

crRNA Array Architectures

Several validated strategies exist for expressing multiple guide RNAs from single transcriptional units, each with distinct advantages for specific applications:

• tRNA-based systems: Utilize endogenous tRNA processing machinery for precise cleavage of polycistronic gRNA transcripts. These systems leverage the endogenous RNase P and RNase Z enzymes to process individual gRNAs from a single transcript, typically achieving high efficiency in dicot and monocot systems [79].
• Ribozyme-mediated processing: Employ hammerhead and hepatitis delta virus ribozymes to flank gRNA units, enabling self-cleavage in the absence of host factors. This approach offers species-independence but may require optimization of ribozyme activity in different plant systems [79].
• Csy4-based systems: Utilize the bacterial RNase Csy4 and its recognition sequence to process gRNA arrays. The Csy4 system provides high processing fidelity but requires co-expression of the Csy4 protein, adding to construct complexity [80].
• Direct synthesis: For viral delivery or transient expression, pre-processed gRNAs can be directly synthesized and delivered as RNA-protein complexes, bypassing the need for processing systems [79].

Table 1: Comparison of Multiplex gRNA Expression Systems

System Type	Processing Mechanism	Advantages	Limitations	Reported Efficiency Range
tRNA-gRNA	Endogenous tRNA processing enzymes	High efficiency in plants; endogenous machinery	Potential tRNA-mediated effects	0-93% editing efficiency [79]
Ribozyme-gRNA	Self-cleaving ribozymes	Species-independent; no protein co-factors required	Variable processing efficiency	0-25% editing efficiency [79]
Csy4-gRNA	Bacterial RNase Csy4	High fidelity processing	Requires Csy4 co-expression	Not specified in results
Individual Pol III	Separate U6/U3 promoters	Predictable expression levels	Size constraints; recombination risk	0-94% editing efficiency [79]

Advanced CRISPR Systems for Multiplexing

Beyond standard Cas9, several engineered CRISPR systems offer enhanced capabilities for multiplex applications:

• Cas12 variants: Cas12a (Cpf1) processes its own crRNA arrays, naturally enabling multiplex editing without additional processing systems. Newer variants like Cas12j and Cas12k offer compact size and different PAM requirements [81].
• Base editors: Enable precise base conversions without double-strand breaks, allowing efficient editing across multiple loci while minimizing collateral damage [80] [81].
• Prime editors: Offer versatile editing capabilities (all 12 possible base-to-base conversions, insertions, and deletions) without requiring donor templates, though efficiency challenges remain for multiplex applications [81].
• Nickase systems: Paired Cas9 nickases targeting opposite strands create staggered double-strand breaks with reduced off-target effects, valuable for precise multiplex editing [80].

Diagram: Workflow for implementing multiplex genome editing strategies in plants, showing key decision points for gRNA expression systems and CRISPR tools.

Complex Trait Loci (CTL) Engineering

The Complex Trait Locus (CTL) approach represents a sophisticated strategy for trait stacking that addresses limitations of random transgene integration. A CTL consists of multiple preselected sites positioned within a small, well-characterized chromosomal region where trait genes can be precisely inserted [82]. This methodology enables flexible trait stacking while ensuring consistent transgene expression and minimizing yield penalties.

CTL Site Selection Criteria

• Genomic conservation: Select regions with conserved haplotypes within germplasm pools (e.g., 34-84% conservation observed in maize inbred lines) [82]
• Low gene density: Target genomic regions with minimal essential genes (4-21 genes per cM in validated maize CTLs) [82]
• High recombination frequency: Prefer regions with high physical-to-genetic distance ratios (0.2-0.6 Mb/cM in maize CTLs) to facilitate future trait separation if needed [82]
• Proximity to existing traits: Leverage regions harboring commercially valuable traits to build upon established loci [82]

Two-Step Trait Integration Protocol

The following protocol outlines the CTL methodology validated in maize [82]:

Step 1: Site-Specific Insertion Landing Pad (SSILP) Integration

• Design SSILP construct (~3 kb) containing FRT recombination sites and selection markers
• Select CRISPR target sites meeting these criteria:
  • At least 2 kb away from any known gene
  • Unique genomic sequence conserved across target inbred lines
  • Unique 200-500 bp flanking sequences
  • Appropriate spacing (0.1-3 cM) within CTL for genetic stacking
• Deliver CRISPR components and SSILP donor via Agrobacterium-mediated transformation
• Regenerate plants and validate precise SSILP integration via PCR and sequencing

Step 2: Trait Gene Integration via Recombinase-Mediated Cassette Exchange (RMCE)

• Transform characterized SSILP lines with trait construct containing corresponding FRT sites
• Induce FLP recombinase expression to catalyze trait cassette exchange
• Select for successful RMCE events using coupled selection systems
• Verify trait integration and expression in T0 generation

Table 2: Complex Trait Locus Performance in Maize

CTL Parameter	CTL1	CTL2	CTL3	CTL4
Chromosomal location	Chr 1	Chr 2	Chr 3	Chr 4
Conservation in SS inbreds	84%	44%	34%	55%
Conservation in NSS inbreds	56%	72%	73%	84%
Gene density (genes/cM)	20	21	4	8
Physical:genetic distance (Mb/cM)	0.4	0.6	0.2	0.2
Number of target sites	30	21	13	12
Genetic span (cM)	4.18	4.28	2.35	3.04
Physical span (Mbp)	2.5	3.2	0.6	0.7

Selection Marker Excision Strategies

Selectable marker genes (SMGs) are essential for transgenic plant selection but raise regulatory and public acceptance concerns. Multiplex CRISPR systems enable efficient SMG excision from established transgenic lines [6].

Marker Excision Protocol

Materials:

• Transgenic plants containing SMG (e.g., DsRED) and gene of interest (GOI)
• CRISPR vector with multiple gRNAs (e.g., 4 gRNAs) targeting SMG flanking regions
• Agrobacterium strain LBA4404
• Plant regeneration medium

Method:

• Design gRNAs targeting both flanking regions of the SMG cassette to induce large deletions (≥10 kb)
• Clone 4 gRNAs into CRISPR vector under appropriate promoters (U6 or U3)
• Transform leaf discs from SMG-containing plants with CRISPR construct
• Regenerate shoots on medium without selection pressure
• Screen regenerated shoots for SMG loss (e.g., loss of red fluorescence for DsRED)
• Confirm SMG excision by PCR amplification across target sites (smaller amplicon indicates deletion)
• Sequence junction sites to verify precise deletion and identify potential small indels
• Validate GOI integrity and expression via PCR and quantitative RT-PCR
• Advance edited plants to T1 generation to segregate out CRISPR components

Expected Results:

• Approximately 20% of regenerated shoots may exhibit SMG loss phenotypes
• About half of these (10% total) typically show molecular confirmation of precise excision
• SMG-free plants display normal growth, flowering, and seed production
• Cas9-free, marker-free transgenic plants can be recovered through segregation in T1 generation [6]

Analytical Methods for Mutation Detection

Accurate detection and quantification of CRISPR edits across multiple loci is crucial for evaluating editing efficiency and characterizing complex genotypes. Multiple methods offer varying levels of sensitivity, throughput, and informational content [71].

Table 3: Benchmarking of Genome Editing Detection Methods

Method	Detection Principle	Sensitivity	Throughput	Informational Content	Cost
T7 Endonuclease 1 (T7E1)	Mismatch cleavage	Low to moderate	Medium	Presence of indels	Low
PCR-RFLP	Restriction site disruption	Moderate	Medium	Presence of indels	Low
Sanger Sequencing + ICE/TIDE	Sequence deconvolution	Moderate	Low to medium	Indel spectrum	Low to medium
PCR-Capillary Electrophoresis/IDAA	Fragment size separation	High	High	Indel sizes	Medium
Droplet Digital PCR (ddPCR)	Allele-specific probes	Very high	High	Specific alleles	High
Targeted Amplicon Sequencing (AmpSeq)	High-throughput sequencing	Highest	Highest	Complete mutation profile	Highest

Recommended Workflow for Multiplex Editing Analysis

• Initial screening: Use PCR-CE/IDAA or ddPCR for rapid efficiency assessment across multiple targets
• Detailed characterization: Employ targeted amplicon sequencing for comprehensive mutation profiling
• Structural variation detection: Implement long-read sequencing (PacBio, Nanopore) to identify large deletions, rearrangements, or complex mutations missed by short-read technologies [79]
• Off-target assessment: Whole genome sequencing remains the gold standard for identifying unintended edits, though targeted approaches to predicted off-target sites can provide a cost-effective alternative

Tissue Culture Integration and Delivery Methods

Effective integration of multiplex editing with plant tissue culture requires optimization of delivery and regeneration protocols. Recent advances in in-planta transformation methods offer alternatives to traditional tissue culture approaches [83].

Delivery Method Selection Guide

• Agrobacterium-mediated transformation: Remain the most efficient and widely used method for stable integration; optimal for embryonic tissues, meristems, and floral parts [83]
• Biolistic delivery: Effective for species recalcitrant to Agrobacterium transformation; enables direct delivery of ribonucleoprotein complexes [83]
• Virus-based systems: Offer high replication and spread of editing components but limited cargo capacity and potential biosafety concerns [79]
• Nanoparticle-mediated delivery: Emerging approach that enables direct delivery of editing reagents while avoiding DNA integration [83]
• Floral dip: Simplified in-planta method effective for Arabidopsis and adapted for some crops; minimal tissue culture requirements [83]

Tissue Culture Workflow for Multiplex Editing

Pre-culture Phase (7-14 days)

• Sterilize explants (seeds, immature embryos, or meristems)
• Pre-culture on appropriate medium to enhance transformation competence

Transformation Phase (2-3 days)

• Inoculate with Agrobacterium carrying multiplex CRISPR construct (OD600 = 0.5-0.8)
• Co-cultivate for 2-3 days in dark conditions

Selection and Regeneration Phase (4-16 weeks)

• Transfer to selection medium containing appropriate antibiotics
• Subculture every 2-3 weeks to fresh selection medium
• Transfer developing shoots to rooting medium

Molecular Analysis Phase

• Extract genomic DNA from regenerated plantlets
• Perform initial PCR screening for presence of transgene
• Conduct deep amplicon sequencing to characterize editing efficiency and heterogeneity across targets

Diagram: Tissue culture and delivery workflow for multiplex genome editing, showing key transformation and regeneration stages with delivery method options.

The Scientist's Toolkit: Essential Research Reagents

Table 4: Key Research Reagents for Multiplex Genome Editing

Reagent/Category	Specific Examples	Function/Application	Considerations
Cas Effectors	SpCas9, LbCas12a, Cas12j	DNA cleavage; different PAM specificities	Size, PAM requirements, specificity
Promoters for gRNAs	AtU6-26, OsU3, GmU6	Drive gRNA expression; species-specific optimization	Expression level, species compatibility
Promoters for Cas	35S, Ubiquitin, EFL	Drive Cas expression; constitutive or tissue-specific	Expression level, toxicity, cell type specificity
Processing Systems	tRNA, Ribozymes, Csy4	Process polycistronic gRNA arrays	Efficiency, species-dependence, additional components
Delivery Vectors	pRIG, pBYR2eFa, pCAS9-TPC	Agrobacterium binary vectors	Size constraints, replication origin, selection markers
Selection Markers	DsRED, NPTII, HPT	Identify transformed tissues	Visual vs. antibiotic selection; excision capability
Modular Cloning Systems	Golden Gate, MoClo	Assembly of multiple gRNA arrays	Standardization, efficiency, library compatibility

Troubleshooting and Optimization

Low Editing Efficiency Across Multiple Targets

• Verify gRNA expression using RT-PCR
• Optimize promoter combinations for different gRNAs
• Test Cas9 codon optimization for specific species
• Evaluate temperature conditions during tissue culture

Somatic Chimerism in Regenerated Plants

• Extend selection period to favor fully edited cells
• Implement secondary regeneration from putative edited tissues
• Utilize early screening methods to identify uniform editors
• Consider meristem-based transformation to reduce chimerism

Structural Rearrangements at Target Sites

• Implement long-range PCR to detect large deletions
• Use paired gRNAs with appropriate spacing (100 bp-10 kb)
• Apply karyotyping or optical mapping for chromosomal abnormalities

Transgene Silencing

• Include introns in expression cassettes
• Utilize matrix attachment regions (MARs) to buffer position effects
• Consider insulators to prevent promoter interference

Multiplex CRISPR editing provides an unprecedented platform for engineering complex polygenic traits in plants. The strategies outlined here enable researchers to address genetic redundancy, stack multiple traits, and accelerate crop improvement programs. As these tools continue to evolve, integration with emerging technologies like artificial intelligence for gRNA design, machine learning for outcome prediction, and novel delivery methods will further enhance the precision and efficiency of multiplex plant genome engineering [79]. The ongoing development of user-friendly, scalable computational workflows for gRNA design, construct assembly, and mutation analysis will be crucial for widespread adoption across diverse crop species [79].

Successful implementation requires careful consideration of vector architecture, delivery methods, and analytical approaches tailored to specific plant systems and experimental goals. By addressing both technical and practical challenges in multiplex editing, researchers can fully leverage this transformative technology to develop next-generation crops with enhanced climate resilience, sustainability, and agricultural productivity.

Minimizing Off-Target Effects and Ensuring Genetic Purity

The integration of CRISPR-based genome editing with plant tissue culture represents a foundational methodology for advancing plant biotechnology. This combination is pivotal for developing crops with enhanced traits, from improved yield and nutritional content to resilience against biotic and abiotic stresses [84]. However, the persistence of off-target effects—unintended genetic modifications at sites other than the intended target—poses a significant risk to genetic purity and the commercial viability of edited plants [85]. Off-target genotoxicity remains a substantial concern that can delay clinical and agricultural translation [85]. Ensuring genetic purity is not merely about achieving the desired edit but confirming that the entire genome remains otherwise unaltered, a non-negotiable standard for both regulatory approval and fundamental research. This Application Note provides a detailed framework of strategies and protocols to minimize off-target effects and verify genetic purity within the context of plant tissue culture and CRISPR-edited plant research, incorporating the latest advancements in AI-guided design and novel delivery systems.

Strategies for Minimizing Off-Target Effects

A multi-faceted approach, encompassing computational design, molecular tool selection, and delivery method optimization, is essential for mitigating off-target effects.

Computational gRNA Design and AI-Guided Optimization

The initial and most critical step in minimizing off-target effects is the strategic design of the guide RNA (gRNA). The principle is to select gRNA sequences with maximal specificity for the target locus and minimal similarity to other genomic regions.

• Leveraging AI-Prediction Models: Artificial intelligence (AI) and machine learning models have revolutionized gRNA design by predicting on-target efficacy and off-target potential with high accuracy [86]. Tools such as DeepCRISPR and CRISPRon leverage deep learning on large-scale experimental datasets to evaluate and rank gRNA candidates based on their predicted performance [86]. These models analyze sequence features, chromatin accessibility, and epigenetic context to provide a reliability score for each gRNA.
• Key Design Parameters: When designing gRNAs, even without specialized AI tools, researchers should prioritize sequences with a high Cutting Frequency Determination (CFD) score and adhere to models like Rule Set 2 for on-target activity prediction [86]. It is crucial to avoid gRNAs with significant homology to other genomic regions, especially those with 1-3 nucleotide mismatches at the 5' end (seed region) or in the PAM-distal region.

Table 1: Key AI Models for gRNA Design and Off-Target Prediction

AI Model/Tool	Primary Function	Key Features	Applicable Plant Systems
DeepSpCas9 [86]	Predicts on-target activity	Uses convolutional neural network (CNN); better generalization across datasets	Human cells; requires validation in plants
CRISPRon [86]	Predicts gRNA efficiency	Trained on a large dataset of 23,902 gRNAs; considers gRNA-DNA binding energy	Human cells; principles applicable to plants
DeepCRISPR [86]	Predicts on/off-target activity	Unsupervised learning; integrates data augmentation and bootstrapping	Human and mouse cells; requires validation in plants
Rule Set 3 [86]	Predicts on-target activity	Incorporates tracrRNA variant influence using LightGBM	Human and mouse cells

Selection of High-Fidelity Cas Variants and Advanced Editors

The choice of the CRISPR-Cas system itself is a major determinant of specificity.

• High-Fidelity Cas Variants: Wild-type Cas9 can tolerate mismatches between the gRNA and DNA, leading to off-target cleavage. Engineered high-fidelity variants such as SpCas9-HF1 and eSpCas9(1.1) contain mutations that reduce non-specific interactions with the DNA backbone, thereby enhancing specificity without significantly compromising on-target activity [84]. Their use is highly recommended for applications requiring high genetic purity.
• Novel AI-Designed Editors: Beyond naturally derived systems, AI is now being used to design novel gene editors from scratch. For instance, OpenCRISPR-1 is a Cas9-like effector protein designed with large language models, which has demonstrated comparable or improved activity and specificity relative to SpCas9, despite being 400 mutations away from any natural sequence [76]. This represents a new frontier in obtaining highly specific editing tools.
• Base and Prime Editors: For precise nucleotide changes without double-strand breaks (DSBs), base editors (BEs) and prime editors (PEs) are superior choices. Base editors facilitate the direct, irreversible conversion of one DNA base into another (e.g., C•G to T•A), while prime editors use a reverse transcriptase to "write" genetic information directly into a target locus using a prime editing guide RNA (pegRNA) [86] [84]. Since these systems do not rely on the error-prone non-homologous end joining (NHEJ) pathway, they drastically reduce the introduction of indels at both on-target and off-target sites.

Optimization of Delivery Methods

The format and method of delivering CRISPR components into plant cells significantly influence off-target profiles.

• Ribonucleoprotein (RNP) Complex Delivery: Delivering pre-assembled complexes of Cas9 protein and gRNA, known as RNPs, is a highly effective strategy [84]. RNPs have a short intracellular lifespan, which limits the window of time for off-target activity. This method also avoids the persistent expression of CRISPR components that occurs with plasmid DNA (pDNA) delivery, which is a major contributor to off-target effects [87].
• Advanced Delivery Platforms: Microfluidic delivery systems, such as the Droplet Cell Pincher (DCP), show great promise for enhancing specificity. The DCP platform uses droplet microfluidics and cell mechanoporation to achieve highly efficient, rapid delivery of CRISPR RNPs into the nucleus. This method has been shown to outperform electroporation in efficiency and can reduce cellular stress, potentially leading to a cleaner editing profile [87].
• Agrobacterium-Mediated Transformation Optimization: For stable plant transformation, Agrobacterium tumefaciens concentration and infection duration must be optimized. A study in Fraxinus mandshurica established an effective editing system by optimizing the OD600 of the bacterial suspension, a critical step for balancing transformation efficiency and cell viability [4].

The following diagram illustrates the integrated multi-layered strategy for minimizing off-target effects, connecting computational design with molecular and delivery optimizations.

Experimental Protocols for Plant Tissue Culture and CRISPR Editing

The following protocols detail the key steps for regenerating genome-edited plants while minimizing off-target risks.

Protocol 3.1: OptimizedAgrobacterium-Mediated Transformation of Plant Explants

This protocol is adapted from the establishment of a CRISPR/Cas9 system in Fraxinus mandshurica and is applicable to many plant species [4].

Materials: Sterile plant explants (e.g., embryos, leaf disks), Agrobacterium tumefaciens strain EHA105 harboring the CRISPR vector, Woody Plant Medium (WPM) solid and liquid media, appropriate antibiotics, acetosyringone.

• Vector Construction: Clone selected high-specificity gRNAs into a CRISPR binary vector (e.g., pYLCRISPR/Cas9P35S-N). Verify constructs via colony PCR and sequencing.
• Plant Material Preparation: Aseptically germinate embryos or prepare explants. Culture on solid WPM medium for 7 days to obtain sterile plantlets [4].
• Agrobacterium Culture Preparation:
  • Inoculate a single colony of the engineered Agrobacterium into LB medium with appropriate antibiotics.
  • Grow overnight at 28°C with shaking until the OD600 reaches a critical range of 0.5 to 0.8.
  • Pellet bacteria by centrifugation (1500 g for 10 min) and resuspend in liquid WPM medium supplemented with 200 µM acetosyringone [4].
• Co-cultivation:
  • Immerse explants in the Agrobacterium suspension for 15-30 minutes.
  • Blot dry and transfer to solid co-cultivation medium. Incubate in the dark at 23-25°C for 2-3 days.
• Selection and Regeneration:
  • Transfer explants to selection medium (e.g., WPM with antibiotics like kanamycin at a pre-determined lethal concentration and a bacteriostatic agent to suppress Agrobacterium).
  • Subculture surviving explants to fresh selection medium every 2-3 weeks to encourage shoot formation.
• Rooting and Acclimatization: Once shoots develop, transfer to rooting medium. After a root system is established, transfer plantlets to soil in a controlled environment for acclimatization.

Protocol 3.2: Ribonucleoprotein (RNP) Delivery via Microfluidic Mechanoporation

For cell types amenable to protoplasting, RNP delivery via systems like the Droplet Cell Pincher (DCP) offers high efficiency and reduced off-target effects [87].

Materials: Plant protoplasts, Cas9 Nuclease protein, synthesized sgRNA, Droplet Cell Pincher (DCP) microfluidic device, FITC-dextran for efficiency validation, culture media.

• RNP Complex Assembly: Complex purified Cas9 protein (e.g., 10 µM) with sgRNA at a molar ratio of 1:2 to 1:5. Incubate at 25°C for 10-15 minutes to form the RNP complex.
• Sample Preparation: Mix protoplast suspension (e.g., 2 × 10^7 cells/mL) with the assembled RNP complexes.
• DCP Processing:
  • Load the cell/RNP mixture and oil into the DCP microfluidic device.
  • Pump the mixture to generate uniform droplets containing cells and RNPs.
  • Accelerate droplets with a sheath oil flow and guide them through a single microscale constriction. This induces transient membrane permeabilization, allowing convective RNP internalization [87].
• Post-Processing Culture: Collect processed droplets and break the emulsion to recover protoplasts. Culture the protoplasts in appropriate regeneration media to allow for cell wall formation and subsequent callus induction and plant regeneration.

Protocols for Verifying Genetic Purity

Rigorous screening is mandatory to confirm on-target editing and the absence of off-target mutations.

Protocol 4.1: Comprehensive Off-Target Assessment

Materials: Genomic DNA from edited and wild-type control plants, PCR reagents, next-generation sequencing (NGS) library preparation kit, primers for on-target and predicted off-target sites.

• In Silico Off-Target Site Prediction: Use the CFD score or an AI-based tool (see Table 1) to generate a list of potential off-target sites in the reference genome. Include sites with up to 5 nucleotide mismatches and indels [86].
• Amplicon Sequencing of Target Regions:
  • Design PCR primers to amplify the on-target locus and all predicted high-risk off-target sites (typically 10-20 sites).
  • Perform PCR on genomic DNA from multiple independent edited lines and a wild-type control.
  • Prepare NGS libraries from the amplicons and sequence at high coverage (>5000x).
• Data Analysis: Align sequencing reads to the reference genome. Use variant calling software to detect insertions, deletions, and single nucleotide variants (SNVs) at each site. The frequency of indels at the on-target site confirms editing efficiency, while the absence of significant variant frequencies above the sequencing error rate (e.g., >0.1%) at off-target sites confirms specificity.

Table 2: Key Reagents for Off-Target Analysis

Reagent / Tool	Function	Application Note
High-Fidelity DNA Polymerase	Amplification of on/off-target loci for sequencing	Essential for error-free PCR to avoid false positives during variant calling.
NGS Library Prep Kit	Preparation of amplicon sequencing libraries	Enables high-throughput, deep sequencing of multiple target sites simultaneously.
CFD Score / AI Prediction Tool	In silico prediction of potential off-target sites	Informs the design of primers for the most likely off-target loci, making screening efficient [86].
Variant Caller Software	Bioinformatics detection of mutations from NGS data	Requires sensitive parameters to detect low-frequency variants but with strict filtering to control false discovery.

Protocol 4.2: Screening for Homogeneity and Chimerism in Regenerated Plants

A key challenge in plant tissue culture is ensuring that regenerated plants are derived from a single edited cell and are not chimeric.

Materials: Tissue culture media, DNA extraction kit, PCR reagents, materials for histological analysis.

• Clustered Bud System for Homozygous Plant Induction: To screen out chimeras, develop a clustered bud system. Subculture and proliferate transformed growing points on media supplemented with cytokinins. Screen multiple buds from a single original growing point; a high percentage (e.g., 18% as reported in Fraxinus mandshurica) of edited buds indicates a non-chimeric origin [4].
• Molecular Genotyping of Individual Segregants:
  • For T0 plants, perform initial genotyping by sequencing the target locus from bulk leaf DNA.
  • To confirm homogeneity, perform single-cell genotyping or genotype multiple individual progeny (T1 generation) from a self-pollinated T0 plant. The segregation of a single edit pattern in the T1 generation is strong evidence that the T0 plant was a non-chimeric homozygous or heterozygous plant.
• Phenotypic Confirmation: Where possible, correlate the genotype with the expected phenotype (e.g., drought tolerance in FmbHLH1 knockout lines) under controlled conditions to confirm the functional outcome of the intended edit [4].

The following workflow outlines the critical path from initial tissue culture to the confirmation of a genetically pure, edited plant line.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Reagents and Kits for CRISPR-Cas Plant Tissue Culture Workflows

Reagent / Kit	Function	Specific Example / Note
High-Fidelity Cas9 Expression Vector	Stable expression of CRISPR nuclease in plant cells.	Vectors like pYLCRISPR/Cas9P35S-N are optimized for plant expression and multiplex gRNA cloning [4].
Chemically Synthesized sgRNA	For RNP complex assembly.	Offers high purity and avoids the need for cloning; ideal for microfluidic delivery [87].
Recombinant Cas9 Protein	For RNP complex assembly.	In-house production is possible with high enzymatic activity, comparable to commercial standards [88].
Plant Tissue Culture Media	Support regeneration of transformed cells.	Woody Plant Medium (WPM) is effective for many tree species; Murashige and Skoog (MS) for others [4].
Selection Antibiotics	Selection of transformed plant cells.	Kanamycin is common; the optimal lethal concentration must be determined empirically for each species [4].
NGS Amplicon-Seq Kit	High-sensitivity off-target detection.	Kits from Illumina or Thermo Fisher are standard for preparing libraries from on/off-target amplicons.
Positive Control gRNA Kit	Transfection and editing efficiency control.	Species-specific positive controls (e.g., for human, mouse) are crucial for optimization [89].

Within the framework of plant tissue culture for CRISPR-based research, achieving precise gain-of-function (GOF) mutations represents a frontier for crop improvement. While traditional CRISPR/Cas9 systems create loss-of-function mutations via double-strand breaks, advanced technologies like Base Editing and CRISPR Activation (CRISPRa) enable more subtle and powerful manipulations. Base editing allows for the direct, irreversible conversion of one base pair to another at a DNA target without requiring double-strand breaks, facilitating the creation of novel alleles and traits. In parallel, CRISPRa employs a deactivated Cas9 (dCas9) fused to transcriptional activators to upregulate endogenous gene expression without altering the underlying DNA sequence. This Application Note details optimized protocols and reagent solutions for implementing these advanced systems, with a particular emphasis on overcoming the challenges associated with recalcitrant species through robust tissue culture methods.

Advanced Genome Editing Systems: Mechanisms and Components

Base Editing Systems

Base editing systems are fusion proteins that combine a catalytically impaired Cas nuclease with a nucleobase deaminase enzyme. They mediate targeted point mutations in genomic DNA without inducing double-strand breaks, significantly reducing unwanted indel mutations [90].

The following table summarizes the core components and properties of the primary base editing systems.

Table 1: Overview of Major Base Editing Systems

System Type	Core Components	Base Conversion	Key Features and Optimized Versions
Cytosine Base Editor (CBE)	nCas9 (D10A) + Cytidine Deaminase + UGI (Uracil Glycosylase Inhibitor)	C•G to T•A	- CBE4max: Incorporates two UGIs and optimized nuclear localization signals (NLS), achieving efficiencies up to 89% [90].- evoFERNY-BE4max: An evolved deaminase with high activity at GC-rich sites [90].
Adenine Base Editor (ABE)	nCas9 (D10A) + Engineered Adenine Deaminase (TadA)	A•T to G•C	- ABE7.10: The first widely used ABE, effective in both plants and animals [90].
Glycosylase Base Editor (GBE)	nCas9 (D10A) + Cytidine Deaminase + Uracil DNA Glycosylase	C•G to G•C	- Enables transversion mutations, expanding the range of possible amino acid changes [90].

The mechanism of CBE systems serves as a representative example. The sgRNA directs the base editor complex to the target genomic locus. The catalytically impaired Cas9, known as nickase Cas9 (nCas9), "unzips" the DNA and exposes a single-stranded DNA R-loop. The fused cytidine deaminase enzyme then acts on a specific window of bases within this single-stranded region, converting cytidine (C) to uridine (U). The subsequent cellular DNA repair machinery recognizes the U as a T, and the complementary strand is nicked and repaired to incorporate an A. The UGI component is critical as it inhibits base excision repair pathways that would otherwise remove the U and revert the change, thereby enhancing editing efficiency [90].

CRISPR Activation (CRISPRa) Systems

Unlike base editing, CRISPRa is designed for transcriptional regulation. It uses a catalytically dead Cas9 (dCas9) that binds to DNA without cutting it. This dCas9 is fused to transcriptional activation domains, which recruit the plant's native transcription machinery to initiate or enhance gene expression from the target locus [59].

This system is particularly valuable for studying gene families with functional redundancy, where knocking out a single gene may not yield a phenotypic change due to compensation by homologous genes. CRISPRa allows for the upregulation of one or multiple genes to decipher their function and create GOF traits, such as enhanced disease resistance [59]. Successful applications include:

• Upregulating the SlPR-1 and SlPAL2 genes in tomato, leading to enhanced defense against bacterial infection and increased lignin accumulation [59].
• Employing a dCas9–6×TAL-2×VP64 system in Phaseolus vulgaris hairy roots to achieve a 6.97-fold increase in expression of the Pv-lectin defense gene [59].

Experimental Protocols

The successful application of these advanced editing tools is contingent upon efficient delivery and regeneration systems. The following protocols outline a highly efficient protoplast-based transfection and regeneration method, as well as a nodal culture system for recalcitrant species.

Protocol 1: Protoplast Regeneration and Transfection for Base Editing

This optimized five-stage protocol for Brassica carinata achieves a regeneration frequency of up to 64% and a transfection efficiency of 40% using a GFP marker, making it ideal for DNA-free delivery of base editing ribonucleoproteins (RNPs) [1].

Workflow Overview:

Detailed Methodology:

• Plant Material Preparation:
  • Source: Use fully expanded leaves from 3- to 4-week-old in vitro seedlings [1].
  • Sterilization: Seeds are surface-sterilized by soaking in 15% (v/v) calcium hypochlorite for 20 minutes, followed by thorough rinsing with sterile water [1].

• Protoplast Isolation:

  • Finely slice leaves and incubate in plasmolysis solution (0.4 M mannitol, pH 5.7) for 30 min in the dark [1].
  • Digest with an enzyme solution containing 1.5% (w/v) cellulase Onozuka R10 and 0.6% (w/v) Macerozyme R10 in 0.4 M mannitol for 14–16 hours in the dark with gentle shaking [1].
  • Filter the protoplast suspension through a 40 µm nylon mesh and purify by centrifugation (100 × g, 10 min) in W5 solution [1].
  • Adjust protoplast density to 400,000–600,000 cells/mL using 0.5 M mannitol [1].

• Protoplast Transfection:

  • Mix the protoplast suspension with an equal volume of 2.8% sodium alginate solution [1].
  • For transfection, incubate protoplasts with pre-assembled Base Editor or CRISPRa RNPs/complexes in a PEG solution (e.g., 40% PEG-4000) [1].
  • Pipette 600 µL of the mixture onto calcium-agar plates to form embedded layers [1].

• Multi-Stage Regeneration Culture: The following table details the media regime critical for success. Table 2: Five-Stage Protoplast Culture Media for Efficient Regeneration [1]

Stage	Medium Name	Key Components & Purpose	Hormonal Ratio (Auxin:Cytokinin)	Culture Duration
Stage 1	MI	High auxins (NAA, 2,4-D) for cell wall formation	High Auxin	7-10 days
Stage 2	MII	Lower auxin for active cell division	Lower Auxin	14 days
Stage 3	MIII	High cytokinin for callus growth & shoot induction	High Cytokinin	14-21 days
Stage 4	MIV	Very high cytokinin for shoot regeneration	Very High Cytokinin	Until shoot emergence
Stage 5	MV	Low BAP and GA₃ for shoot elongation	Low/No PGR	Until shoots are 2-3 cm

Protocol 2: Nodal Culture for Recalcitrant Horticultural Crops

For species resistant to protoplast regeneration, nodal culture provides a robust alternative for regeneration and transformation.

Workflow Overview:

Detailed Methodology:

• Explant Preparation and Sterilization:
  • Collect 1–2 cm long immature nodal segments [20].
  • Clean with liquid detergent (Tween 20) and rinse with distilled water [20].
  • Immerse in a fungicide-bactericide solution (carbendazim and streptocycline) for 20 minutes [20].
  • Surface sterilize with 70% ethanol for 5 minutes, followed by 0.8–1.0% sodium hypochlorite for 20 minutes [20].

• Shoot Regeneration:

  • Inoculate sterilized nodal explants on MS or DKW medium [20].
  • Supplement the medium with a combination of auxin (0.01–2 mg/L) and cytokinin (0.4–4 mg/L) to promote shoot formation [20].
  • Maintain cultures at 25 ± 2°C with a 16-hour photoperiod. Shoot regeneration typically occurs within 4–8 weeks [20].

• Genetic Transformation:

  • Use the nodal segments for Agrobacterium tumefaciens or Rhizobium rhizogenes-mediated delivery of base editor or CRISPRa constructs [91] [20].
  • Alternatively, meristematic cells in the nodes can be targeted for in planta transformation to minimize tissue culture steps [91].

• Rooting and Acclimatization:

  • Transfer regenerated shoots to half-strength WPM or full-strength DKW media containing 0.1–2 mg/L of auxin for root induction [20].
  • Root development is typically observed within 4 weeks [20].
  • Acclimatize plantlets in a peat:perlite (2:1) mixture under high humidity (>85%) before gradual transfer to greenhouse conditions [20].

The Scientist's Toolkit: Research Reagent Solutions

The table below lists essential reagents and their functions for implementing the protocols described in this note.

Table 3: Essential Research Reagents for Base Editing and CRISPRa Workflows

Reagent / Material	Function / Application	Example Specifications / Notes
Cellulase "Onozuka" R10	Plant cell wall digestion for protoplast isolation [1].	Used at 1.5% (w/v) in enzyme solution.
Macerozyme R10	Pectin degradation for protoplast isolation [1].	Used at 0.6% (w/v) in combination with Cellulase.
Sodium Alginate	For embedding protoplasts in a thin layer, supporting early development [1].	Used at 2.8% (w/v) mixed with protoplasts.
Polyethylene Glycol (PEG)	Mediates the delivery of RNPs or DNA into protoplasts (PEG-mediated transfection) [1].	Typically PEG-4000, used at 40% concentration.
Mannitol	Provides osmotic stability to protoplasts and enzyme solutions [1].	Used at 0.4 M concentration.
Base Editor RNP	Pre-assembled complex of purified nCas9-deaminase protein and sgRNA for DNA-free base editing.	Can be delivered via PEG transfection to protoplasts.
dCas9-Activator Construct	Plasmid or RNP for CRISPRa; dCas9 fused to transcriptional activators (e.g., VP64, TAL) [59].	For stable transformation or transient expression.
Murashige and Skoog (MS) Medium	Basal nutrient medium for plant tissue culture [1] [20].	Used full- or half-strength, often with vitamins.
Driver & Kuniyuki (DKW) Medium	Woody Plant Medium, often superior for tree and recalcitrant species [20].	Used for shoot and root induction in nodal culture.
Plant Growth Regulators (PGRs)	Critical for directing organogenesis (e.g., NAA, 2,4-D, BAP, Zeatin) [1] [20].	Specific combinations and ratios are stage-dependent.

Base editing and CRISPRa technologies represent a paradigm shift in plant biotechnology, moving beyond gene knockouts to enable precise single-nucleotide changes and targeted gene activation. Their successful implementation, however, hinges on robust tissue culture and regeneration systems. The protocols detailed here—ranging from the high-efficiency protoplast system for amenable species to the nodal culture technique for recalcitrant crops—provide a practical roadmap for researchers. By leveraging these advanced editing tools alongside optimized regeneration protocols, scientists can accelerate the development of crops with enhanced resilience, yield, and nutritional quality, paving the way for a new era in molecular plant breeding.

Ensuring success: Analytical methods and tool comparison for edited plants

In plant tissue culture and CRISPR-edited plant research, molecular validation is a critical, multi-stage process for confirming successful genome modifications. Following the delivery of CRISPR-Cas9 components into plant cells and subsequent regeneration of whole plants from cultured tissues, researchers must employ a suite of molecular techniques to detect, quantify, and characterize the induced genetic changes. This process begins with initial screening to identify potentially edited individuals from a population of regenerants and progresses to precise quantification of editing efficiency and detailed analysis of mutation profiles. The selection of appropriate validation methods is paramount, as it directly impacts the accuracy, reliability, and reproducibility of research outcomes. These techniques must be capable of detecting a wide spectrum of mutations—from single nucleotide changes to large insertions or deletions—often within the complex context of polyploid plant genomes and heterogeneous cell populations derived from tissue culture. The choice of method typically involves balancing factors such as sensitivity, throughput, cost, and technical requirements, with different approaches being better suited to specific stages of the research pipeline, from initial screening to final characterization of homozygous mutant lines.

Multiple molecular techniques have been adapted or developed specifically for validating CRISPR-Cas9 edits in plants, each with distinct operational principles, advantages, and limitations. These methods can be broadly categorized into enzyme-based mismatch detection assays, sequencing-based approaches, and electrophoresis-based fragment analysis methods.

Enzyme mismatch cleavage assays, such as the T7 Endonuclease I (T7E1) assay and Surveyor nuclease assay, function by recognizing and cleaving DNA heteroduplexes formed when wild-type and mutated DNA strands hybridize. The T7E1 assay begins with PCR amplification of the target region from genomic DNA of potentially edited plants. The resulting amplicons, which contain a mixture of wild-type and mutant sequences in edited samples, are denatured and reannealed to form heteroduplexes at mismatch sites corresponding to mutation locations. The T7E1 enzyme then cleaves these heteroduplexes, and the digestion products are visualized using agarose gel electrophoresis. The ratio of cleaved to uncleaved DNA bands provides an estimate of editing efficiency [92]. While these enzyme-based methods are cost-effective and provide same-day results without requiring specialized equipment, they have significant limitations: they cannot identify the specific sequence changes, are less sensitive to low-frequency edits and single-nucleotide changes, and can yield false positives from naturally occurring polymorphisms in plant genomes [92] [39].

Sequencing-based approaches offer the highest level of detail by directly determining the DNA sequence at target loci. Sanger sequencing, when combined with decomposition algorithms like Tracking of Indels by Decomposition (TIDE) or Inference of CRISPR Edits (ICE), can quantify editing efficiencies from a mixed population of cells by comparing sequencing chromatograms from edited samples to wild-type controls and computationally decomposing the complex signals into specific indel combinations and frequencies [71] [92]. Next-generation sequencing methods, particularly targeted amplicon sequencing (AmpSeq), provide the most comprehensive analysis by sequencing thousands of individual DNA molecules from a single sample, enabling highly sensitive detection of low-frequency mutations, precise quantification of editing efficiency, and complete characterization of the spectrum of induced mutations in a heterogeneous plant population [71]. While AmpSeq is considered the "gold standard" for accuracy and sensitivity, its routine use can be limited by higher costs, longer turnaround times, and the need for specialized bioinformatics expertise and computational resources [71].

Electrophoresis-based methods detect edits through changes in DNA fragment size or mobility. PCR-restriction fragment length polymorphism (PCR-RFLP) exploits the frequent destruction or creation of restriction enzyme sites by CRISPR-induced mutations, allowing differentiation between edited and wild-type alleles through restriction digestion and fragment analysis [71]. PCR-capillary electrophoresis/InDel detection by amplicon analysis (PCR-CE/IDAA) separates fluorescently labeled PCR products by size using capillary electrophoresis, providing high-resolution detection of different indel mutations and their relative frequencies based on fragment sizes [71]. Droplet digital PCR (ddPCR) enables absolute quantification of editing efficiency by partitioning a PCR reaction into thousands of nanoliter-sized droplets and counting positive and negative reactions for mutant and wild-type alleles, offering exceptional sensitivity and precision without requiring standard curves [71].

Table 1: Comparison of Key CRISPR Validation Techniques for Plant Research

Method	Detection Principle	Sensitivity	Information Obtained	Throughput	Relative Cost	Best Use Cases
T7E1 Assay	Enzyme mismatch cleavage	Moderate (~5%)	Estimated efficiency, no sequence detail	Low-medium	$	Initial screening, rapid validation
Sanger + TIDE/ICE	Sequencing & deconvolution	Moderate (~5%)	Indel types, frequencies	Low-medium	$$	Efficiency quantification, mutation profiling
Targeted Amplicon Seq	High-throughput sequencing	High (<0.1%)	Complete mutation spectrum, precise quantification	High	$$$	Gold-standard validation, characterization
PCR-RFLP	Restriction site alteration	Moderate (~5%)	Efficiency (site-dependent)	Low	$	Rapid validation when restriction site affected
PCR-CE/IDAA	Capillary electrophoresis	High (1-2%)	Indel sizes, frequencies	Medium	$$	Multiplexing, precise fragment analysis
ddPCR	Digital quantification	High (0.1-1%)	Absolute quantification	Medium	$$	Sensitive quantification, rare allele detection
qEva-CRISPR	Probe ligation & qPCR	High (0.1-1%)	Quantitative, multiplex capable	High	$$	High-throughput, multiplex target analysis

Detailed Experimental Protocols

T7 Endonuclease I (T7E1) Mismatch Cleavage Assay

The T7E1 assay provides a rapid, cost-effective method for initial screening of CRISPR-Cas9 editing in plant tissues, particularly useful for evaluating multiple sgRNA targets or optimization parameters during preliminary experiments.

Materials and Reagents:

• Plant genomic DNA extraction kit (e.g., TIANGEN Biotech Plant Genomic DNA Kit)
• High-fidelity DNA polymerase (e.g., AccuTaq LA DNA Polymerase)
• T7 Endonuclease I enzyme (available in commercial kits)
• Agarose gel electrophoresis equipment
• PCR purification kit
• Target-specific PCR primers flanking the CRISPR target site

Protocol Steps:

• Genomic DNA Extraction: Isolate high-quality genomic DNA from CRISPR-treated plant tissues and wild-type controls using a standardized plant DNA extraction method. For tissue culture-derived plants, sample approximately 100mg of leaf tissue. Quantify DNA concentration using spectrophotometry and adjust to 20-50ng/μL for PCR [92].

• PCR Amplification: Amplify the target region using high-fidelity DNA polymerase to prevent introduction of polymerase errors that could be misinterpreted as edits. Set up 25-50μL reactions with 1× PCR buffer, 0.2mM dNTPs, 0.5μM forward and reverse primers, 1-2 units polymerase, and 50-100ng genomic DNA template. Use the following cycling conditions: initial denaturation at 95°C for 3min; 35 cycles of 95°C for 30s, primer-specific annealing temperature (typically 55-65°C) for 30s, 72°C for 30-60s (depending on amplicon size); final extension at 72°C for 5min [92].

• PCR Product Purification: Purify amplification products using a PCR purification kit according to manufacturer's instructions. Elute in nuclease-free water or TE buffer and quantify using spectrophotometry.

• Heteroduplex Formation: Denature and reanneal the PCR products to form heteroduplexes between wild-type and mutant strands. Use 100-200ng purified PCR product in a 10μL reaction with 1× NEBuffer 2. Use the following thermal cycler program: 95°C for 5min, ramp down to 85°C at -2°C/sec, then to 75°C at -0.3°C/sec, then to 65°C at -0.3°C/sec, then to 55°C at -0.3°C/sec, then to 45°C at -0.3°C/sec, then to 35°C at -0.3°C/sec, then to 25°C at -0.3°C/sec, and hold at 4°C [92].

• T7E1 Digestion: Add 1μL T7 Endonuclease I (commercially available units) to the heteroduplex reaction and incubate at 37°C for 15-60 minutes. Include a no-enzyme control to assess non-specific degradation.

• Analysis by Gel Electrophoresis: Separate digestion products on a 2-3% agarose gel containing ethidium bromide or SYBR-safe DNA gel stain. Include appropriate DNA size markers. Visualize under UV light and document. Successful editing is indicated by the presence of additional cleavage fragments beyond the expected full-length PCR product. Calculate approximate editing efficiency using the formula: % editing = [1 - (1 - (a + b)/(a + b + c))^0.5] × 100, where c is the intensity of the uncut band, and a and b are the intensities of the cleavage products [92].

Sanger Sequencing with TIDE Analysis

This method combines traditional Sanger sequencing with computational decomposition to quantify editing efficiencies and identify predominant mutation types from bulk PCR products of heterogeneous plant samples.

Materials and Reagents:

• Plant genomic DNA extraction kit
• High-fidelity DNA polymerase
• PCR purification kit
• Sanger sequencing reagents or commercial sequencing service
• TIDE analysis web tool (https://tide.nki.nl)

Protocol Steps:

• DNA Extraction and PCR Amplification: Isolate genomic DNA from putative edited plants and wild-type controls as described in section 3.1. Amplify the target region using high-fidelity polymerase with primers flanking the CRISPR target site. Include a minimum of three biological replicates per sample to ensure statistical reliability [71].

• PCR Product Purification and Sequencing: Purify amplification products and submit for Sanger sequencing using one of the PCR primers. Ensure high-quality sequencing traces with low background noise by following sequencing facility recommendations for sample concentration and purity.

• TIDE Analysis:

  • Access the TIDE web tool and upload the sequencing chromatogram files from both the edited sample and the wild-type control.
  • Set the parameters including the target sequence and the CRISPR cut site location (typically 3-4 base pairs upstream of the PAM sequence).
  • The algorithm decomposes the complex sequencing trace from the edited sample by comparing it to the wild-type reference, identifying the most prevalent indels and their frequencies in the population.
  • Review the quality metrics provided by the software, including the p-value for significance of detected indels and the goodness of fit (R²) of the decomposition model.
  • Export results including the spectrum of identified mutations, their frequencies, and the overall editing efficiency [71] [92].

• Data Interpretation: The TIDE output provides the percentage of edited alleles in the sample population and identifies the specific indel mutations present. For plant tissue culture applications, where chimerism is common in primary regenerants, this method helps estimate the proportion of edited cells and guides selection of plants for further propagation and molecular analysis.

Targeted Amplicon Sequencing (AmpSeq)

Targeted amplicon sequencing provides the most comprehensive analysis of CRISPR editing outcomes by sequencing thousands of individual DNA molecules, enabling detection of low-frequency mutations and complete characterization of the editing spectrum.

Materials and Reagents:

• Plant genomic DNA extraction kit
• High-fidelity DNA polymerase
• Library preparation kit for Illumina platforms
• AMPure XP beads or similar magnetic bead-based purification system
• Quantitation kit for NGS libraries (e.g., Qubit dsDNA HS Assay)
• Illumina sequencing platform or commercial sequencing service

Protocol Steps:

• Experimental Design and Primer Design: Design two-step PCR primers with the target-specific sequence in the first step and Illumina adapter sequences with sample barcodes in the second step. The target amplicon should be 300-500bp, centered on the CRISPR cut site. Include unique dual indices for each sample to enable multiplexing [71].

• Primary PCR Amplification: Perform the first PCR with target-specific primers using high-fidelity polymerase to minimize amplification errors. Use a minimal number of cycles (typically 20-25) to maintain representation while generating sufficient product. Include negative controls to detect contamination.

• Library Preparation and Indexing: Purify primary PCR products using magnetic beads. Perform a second, limited-cycle PCR (typically 8-12 cycles) to add full Illumina adapter sequences with unique dual indices for each sample. This enables multiplexing of multiple samples in a single sequencing run.

• Library Quantification and Pooling: Quantify final libraries using fluorometric methods and pool equimolar amounts of each library based on quantification results. Validate library quality and size distribution using capillary electrophoresis (e.g., Bioanalyzer or TapeStation).

• Sequencing and Data Analysis: Sequence on an appropriate Illumina platform (MiSeq or MiniSeq for smaller studies; NovaSeq for larger projects) to achieve sufficient coverage (>10,000x per sample). Process raw data through a bioinformatics pipeline typically involving: demultiplexing, quality filtering, alignment to reference sequence, and indel calling centered on the expected cut site. Specialized tools like CRISPResso2 are available for precise quantification of editing efficiencies and mutation spectra [71].

Table 2: Research Reagent Solutions for CRISPR Validation

Reagent/Category	Specific Examples	Function in Validation	Application Notes
High-Fidelity Polymerases	AccuTaq LA DNA Polymerase	PCR amplification of target loci	Critical for minimizing polymerase errors that confound edit detection [92]
Commercial Cleavage Kits	GeneArt Genomic Cleavage Detection Kit	T7E1 mismatch cleavage assay	Standardized reagents for reliable enzyme-based detection [93]
NGS Library Prep Kits	Illumina DNA Prep kits	Targeted amplicon sequencing	Enable high-sensitivity mutation detection and quantification [71]
Digital PCR Systems	Droplet digital PCR (ddPCR)	Absolute quantification of edits	Provides high sensitivity (0.1%) without standard curves [71]
Capillary Electrophoresis	PCR-CE/IDAA systems	Fragment analysis for indel detection	High-resolution size detection for indels [71]
Cloning Vectors	TA cloning kits	Sanger sequencing of individual alleles	Enables isolation of individual mutant alleles for characterization

Advanced and Emerging Techniques

Quantitative Evaluation of CRISPR Edits (qEva-CRISPR)

The qEva-CRISPR method represents an advanced approach that combines the principles of multiplex ligation-dependent probe amplification (MLPA) with quantitative PCR to enable highly sensitive, quantitative assessment of editing efficiency at multiple target sites simultaneously.

Principles and Applications: qEva-CRISPR uses short oligonucleotide probes that hybridize to sequences flanking the CRISPR target site. When the target sequence is intact, the probes ligate and form a template for quantitative PCR amplification. Edited alleles containing mutations at the target site prevent proper probe hybridization and ligation, reducing PCR amplification signal proportionally to the editing efficiency. This method is particularly valuable for plant research applications requiring multiplex analysis of several targets or monitoring of potential off-target effects in parallel [39].

Key Advantages:

• Capable of detecting all mutation types, including single nucleotide changes and large deletions
• Sensitivity down to 0.1-1% editing frequency
• Enables simultaneous analysis of multiple targets (on-target and off-target sites) in a single reaction
• Successful for analyzing "difficult" genomic regions with high GC content or repetitive sequences
• Can distinguish between homology-directed repair (HDR) and non-homologous end joining (NHEJ) outcomes [39]

Implementation Considerations: While qEva-CRISPR offers significant advantages for comprehensive editing analysis, it requires careful probe design and optimization for each target site. The method involves multiple steps including probe hybridization, ligation, and quantitative PCR, necessitating rigorous optimization of reaction conditions. However, once established, it provides a robust platform for high-throughput screening of editing efficiency across multiple targets and samples.

Real-Time PCR-Based Detection Methods

Advanced real-time PCR methods have been developed for specific applications in plant gene editing, particularly for detection of single-nucleotide edits and screening of transgene-free edited plants.

TaqMan Probe-Based Detection: This approach uses allele-specific fluorescent probes to distinguish between wild-type and edited sequences in real-time PCR. For edited tomato plants with single-nucleotide deletions, researchers developed a multiplex TaqMan real-time PCR system using dual fluorescently labeled probes simultaneously targeting edited and unedited sequences. This method enabled sensitive detection down to 0.1% of edited lines in mixed samples and provided a reliable approach for quality control and regulatory compliance [94].

Applications in Transgene-Free Editing: For plants edited using transient expression or ribonucleoprotein (RNP) delivery methods, real-time PCR serves a dual purpose: verifying successful editing and confirming the absence of CRISPR transgenes in the final plants. This is particularly important for regulatory compliance and public acceptance. The method typically involves initial screening using rapid techniques like LAMP (loop-mediated isothermal amplification) or conventional PCR targeting the Cas9 transgene, followed by verification of the specific edit using real-time PCR with allele-specific probes [94] [95].

Implementation Strategy for Plant Research

Establishing an effective molecular validation pipeline for plant CRISPR research requires strategic planning aligned with research objectives and resource constraints. A tiered approach typically provides the optimal balance of thoroughness and efficiency.

Initial Screening Phase: For rapid assessment of multiple sgRNA designs or optimization of delivery parameters, implement high-throughput, cost-effective methods like T7E1 or PCR-RFLP. These approaches allow efficient screening of large plant populations regenerated from tissue culture to identify potentially edited individuals for further analysis. At this stage, focus on identifying samples with detectable editing activity rather than precise quantification [71] [92].

Efficiency Quantification Phase: For confirmed edited plants, progress to more quantitative methods such as Sanger sequencing with TIDE/ICE analysis or digital PCR. These approaches provide reliable quantification of editing efficiency and preliminary information about mutation types, helping prioritize lines for further propagation and phenotypic analysis. This stage is particularly important for polyploid plant species, where editing efficiency across multiple homeologs must be assessed [71].

Comprehensive Characterization Phase: For final validation and detailed molecular characterization of lead lines, employ targeted amplicon sequencing (AmpSeq) or qEva-CRISPR. These methods provide complete information about the mutation spectrum, including precise sequence changes, mutation frequencies, and potential unpredicted edits. This comprehensive analysis is essential for publication-quality data and for regulatory compliance when developing commercial crop varieties [71] [39].

Method Selection Considerations: When designing a validation strategy, consider polyploidy in many plant species, which complicates editing detection and quantification. Choose methods capable of distinguishing between edited and non-edited homeologs and accurately quantifying editing efficiency in complex genomic backgrounds. For vegetatively propagated plants or those with long life cycles, prioritize methods that efficiently identify homogeneous edited lines without chimerism, such as careful sampling of multiple tissue types followed by sensitive detection methods [71] [95].

The establishment of a robust molecular validation pipeline is fundamental to successful plant genome editing research. By implementing appropriate techniques at each stage of the workflow—from initial screening to comprehensive characterization—researchers can reliably detect, quantify, and characterize CRISPR-induced mutations, accelerating the development of improved crop varieties through precision genome editing.

Phenotypic screening serves as a critical bridge between genetic modifications, such as those introduced by CRISPR-based genome editing, and their functional outcomes in plant biology. Within the context of plant tissue culture and CRISPR-edited plant research, this process enables researchers to identify and characterize desirable traits ranging from cellular-level changes in somatic tissues to complex whole-plant characteristics [96] [97]. The integration of advanced genome editing technologies with sophisticated phenotypic screening protocols has revolutionized functional genomics and crop improvement strategies, allowing for precise modulation of gene function and the identification of novel genes controlling important agronomic traits [96].

The emergence of CRISPR-Cas systems has provided unprecedented precision in generating genetic diversity for phenotypic screening. While initial applications focused primarily on creating loss-of-function mutations through knockouts, recent advancements have expanded to include gain-of-function approaches using CRISPR activation (CRISPRa) systems [96]. These technological developments are particularly valuable for plant research, where genetic redundancy often obscures phenotypic outcomes from single gene knockouts [97]. This application note details comprehensive protocols for implementing phenotypic screening across different biological scales, from initial somatic tissue evaluation to whole-plant trait assessment, specifically tailored for CRISPR-edited plants generated through tissue culture systems.

Phenotypic Screening Platforms and Parameters

Table 1: Comparison of Phenotypic Screening Platforms for CRISPR-Edited Plants

Screening Platform	Tissue/Plant Stage	Key Readout Parameters	CRISPR Editing Approach	Throughput Capacity
Somatic Tissue Screening	Callus, protoplasts, hairy roots	Gene expression (qRT-PCR), metabolite levels, cellular morphology, reporter gene expression	CRISPR knockout, base editing, CRISPRa [96] [97]	High (96/384-well formats)
Tissue Culture-Based Screening	In vitro plantlets, embryos	Regeneration efficiency, somatic embryogenesis, organogenesis, hormone response	CRISPR knockout, CRISPRa (e.g., SlWRKY29) [96]	Medium to High
Whole-Plant Screening	Mature soil-grown plants	Disease resistance [96], architectural traits, yield components, physiological parameters	Multiplexed CRISPR knockout, viral delivery systems [98]	Low to Medium

Table 2: Quantitative Parameters for Phenotypic Assessment Across Developmental Stages

Developmental Stage	Morphological Parameters	Physiological/Biochemical Parameters	Molecular Parameters	Timeline
Somatic Tissues	Callus size, color, texture, embryo formation	Secondary metabolite production, hormone sensitivity, enzyme activity	Transcript levels (fold-change), protein abundance, epigenetic marks	2-8 weeks
In Vitro Plantlets	Root length, shoot height, leaf number, apical dominance	Chlorophyll content, nutrient uptake, stress indicator compounds	Pathway-specific marker gene expression	4-12 weeks
Mature Plants	Plant height, internode length, leaf area, flowering time	Photosynthetic rate, water use efficiency, disease scoring [96]	Biomass allocation, yield components, seed composition	8-24 weeks

Experimental Protocols

Protocol 1: High-Throughput Phenotypic Screening in Somatic Tissues

Purpose: To establish a reliable workflow for early-stage phenotypic screening of CRISPR-edited somatic tissues prior to plant regeneration.

Materials:

• CRISPR-edited callus cultures or protoplasts
• Tissue culture media (appropriate for plant species)
• Laminar flow hood, sterile containers
• RNA/DNA extraction kits
• qRT-PCR equipment and reagents
• Microscopy imaging system

Procedure:

• Initiate CRISPR Editing: Transform plant explants using Agrobacterium-mediated or biolistic methods with your CRISPR construct. For efficient delivery, consider engineered plant viruses like tobacco rattle virus for compact CRISPR systems [98].
• Generate Editing Events: Culture transformed tissues on appropriate selection media for 2-4 weeks to generate independent editing events.
• Molecular Validation: Isolate genomic DNA from sub-samples and perform PCR amplification of target regions. Verify editing efficiency through restriction fragment length polymorphism (RFLP) analysis, sequencing, or T7E1 assay.
• Phenotypic Assessment in Callus:
  • Document visual phenotypes (color, texture, morphology) weekly
  • Quantify growth rates by measuring fresh weight gain
  • For CRISPRa experiments, validate gene activation via qRT-PCR [96]
  • Analyze metabolite profiles if targeting metabolic pathways
• Data Collection: Capture high-resolution images and quantify at least three biological replicates per editing event.
• Selection of Events: Prioritize events showing desired molecular and phenotypic changes for regeneration.

Troubleshooting Tips:

• Low editing efficiency: Optimize sgRNA design and delivery method
• High phenotypic variability: Increase sample size and implement randomized plating
• Poor regeneration: Adjust hormone concentrations in regeneration media

Protocol 2: Whole-Plant Phenotypic Screening for Disease Resistance

Purpose: To evaluate disease resistance phenotypes in regenerated CRISPR-edited plants under controlled conditions.

Materials:

• Regenerated T0 or T1 CRISPR-edited plants
• Appropriate pathogen isolates or inoculum
• Growth chambers or greenhouse facilities
• Disease assessment tools (rating scales, imaging equipment)
• RNA/DNA extraction kits

Procedure:

• Plant Establishment: Acclimate regenerated plants to soil conditions and maintain under controlled environmental settings.
• Molecular Confirmation: Verify heritability of CRISPR edits through genotyping of T1 generation when possible. For transgene-free editing, use systems that employ viral delivery without DNA integration [98].
• Experimental Design: Randomize plants across treatments and replicates. Include appropriate controls (wild-type, empty vector).
• Pathogen Inoculation:
  • Prepare standardized inoculum of target pathogen
  • Apply using appropriate method (spraying, injection, root-dipping)
  • Include mock-inoculated controls
• Disease Assessment:
  • Monitor disease progression daily
  • Quantify using standardized rating scales (e.g., 0-5 for disease severity)
  • Document with photography at regular intervals
  • For CRISPRa-enhanced resistance, compare timing and amplitude of defense gene activation (e.g., SlPR-1, SlPAL2) [96]
• Molecular Analysis:
  • Sample tissue at multiple time points for gene expression analysis
  • Measure defense-related metabolites (e.g., phytoalexins, lignin)
  • For successful CRISPRa applications, confirm sustained upregulation of target defense genes [96]
• Data Analysis: Perform statistical comparisons between edited and control plants for disease parameters and molecular markers.

Troubleshooting Tips:

• Inconsistent infection: Standardize inoculum concentration and environmental conditions
• Escapism: Ensure uniform inoculation coverage
• Variable plant growth: Maintain uniform growing conditions

Workflow Visualization

Figure 1: Comprehensive workflow for phenotypic screening of CRISPR-edited plants across developmental stages.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for Phenotypic Screening

Reagent/Category	Specific Examples	Function/Application	Considerations
CRISPR Delivery Systems	Agrobacterium strains, tobacco rattle virus vectors [98], gold microparticles	Introduction of CRISPR components into plant cells	Size limitations for viral delivery [98], species-specific optimization
CRISPR Nucleases	SpCas9, ISYmu1 [98], dCas9 transcriptional activators [96]	Targeted DNA cleavage or gene regulation	PAM requirements, size constraints for viral vectors [98]
Transcriptional Modulators	dCas9-VP64, dCas9-p65, plant-specific PTAs [96]	Targeted gene activation (CRISPRa) for gain-of-function studies	Strength of activation domain, plant-specific optimization [96]
Tissue Culture Media	Callus induction, somatic embryogenesis, shoot regeneration media	Support growth and development of edited tissues	Species-specific formulations, hormone optimization
Selection Agents	Antibiotics (kanamycin, hygromycin), herbicides	Selection of successfully transformed tissues	Species-specific sensitivity, concentration optimization
Reporter Systems	GUS, GFP, YFP	Visual tracking of transformation and gene expression	Compatibility with imaging systems, minimal physiological disruption
Molecular Analysis Kits	DNA extraction, RNA isolation, qRT-PCR reagents	Molecular validation of edits and expression changes	Compatibility with plant tissues, high-throughput capability

Advanced Applications and Integration

The integration of phenotypic screening with multi-omics approaches represents a powerful strategy for comprehensive functional analysis. CRISPR screens in plants enable systematic functional genomics at an unprecedented scale, allowing researchers to map gene networks and identify key regulators of important traits [97]. For instance, CRISPR knockout screens can generate mutant collections targeting entire gene families, while CRISPRa screens can identify genes that confer desirable traits when overexpressed [96].

Recent advancements in viral delivery systems for CRISPR components, such as the engineered tobacco rattle virus carrying compact CRISPR systems, offer opportunities to overcome transformation bottlenecks in recalcitrant species [98]. These developments are particularly valuable for high-throughput phenotypic screening as they can potentially enable editing in a wider range of plant species without the need for extensive tissue culture optimization.

The future of phenotypic screening in plant research will likely involve increased automation, integration with sensor-based phenotyping technologies, and the implementation of more sophisticated CRISPR systems capable of multiplexed editing and precise transcriptional control. These advancements will accelerate the identification and characterization of genes controlling important agronomic traits, ultimately facilitating the development of improved crop varieties with enhanced productivity, nutritional quality, and resilience to environmental challenges.

The application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in plant biology has revolutionized genome engineering, offering unprecedented precision for crop improvement. Within plant research, a significant bottleneck remains the reliance on tissue culture for regenerating whole plants from edited cells, a process that is often time-consuming, genotype-dependent, and inefficient for many species [2]. The choice of CRISPR system is therefore critical, not only for editing efficiency but also for compatibility with emerging tissue culture-free transformation methods. This application note provides a comparative analysis of the most widely used CRISPR nucleases—Cas9 and Cas12—alongside novel engineered variants, focusing on their operational parameters, optimized protocols for plants, and their role in advancing plant biotechnology by overcoming regeneration limitations.

Comparative Analysis of CRISPR Nucleases

The core components of a CRISPR system include the Cas nuclease and a guide RNA, which form a ribonucleoprotein complex that targets and cleaves specific DNA sequences adjacent to a Protospacer Adjacent Motif (PAM) [99] [100]. The properties of the nuclease determine the system's targeting range, editing outcome, and delivery feasibility.

Table 1: Key Characteristics of Major CRISPR Nucleases in Plant Systems

Nuclease	PAM Sequence	Size (aa)	Cut Type	Guide RNA	Key Advantages	Reported Editing Efficiency in Plants
SpCas9 (Streptococcus pyogenes)	5'-NGG-3' [99]	~1368 [99]	Blunt-ended DSB [101]	sgRNA (crRNA + tracrRNA) [99]	Most widely used; extensive toolkits [102]	Up to 100% in barley (T0) with ZmCas9+13int [102]
SaCas9 (Staphylococcus aureus)	5'-NNGRRT-3' [99]	1053 [99]	Blunt-ended DSB	sgRNA	Small size ideal for viral delivery (AAV) [99]	High efficiency in tobacco, potato, and rice [99]
LbCas12a (Lachnospiraceae bacterium)	5'-TTTV-3' [103]	~1200-1300	Staggered DSB [101]	Short crRNA (42 nt) [103]	Enables multiplexing; useful for GC-rich targets [102]	90% mutant alleles in barley; 86-93% in wheat (T0) [102]
ttLbCas12a Ultra V2 (Engineered)	5'-TTTV-3' [103]	~1200-1300	Staggered DSB	Short crRNA	Enhanced activity & temperature tolerance [103]	20.8% to 99.1% in Arabidopsis T1 plants [103]
hfCas12Max (Engineered)	5'-TN-3' [99]	1080 [99]	Staggered DSB	crRNA	Broad PAM; high fidelity; small size [99]	Under development for therapeutics (e.g., Duchenne muscular dystrophy) [99]
AsCas12f1	5'-YTTN- or NTTR-3' [103]	~400-700 [103] [104]	Staggered DSB	crRNA	One of the smallest Cas nucleases [103]	Poor or no detectable editing in plants [103]

Critical Selection Criteria for Plant Research

• PAM Specificity and Targeting Range: The PAM sequence is a primary determinant of targetable sites. SpCas9's NGG PAM is common in GC-rich genomes, while Cas12a's TTTV PAM is advantageous for targeting AT-rich regions like promoters and introns [102]. Engineered variants like hfCas12Max (TN PAM) and ScCas9 (NNG PAM) offer broader targeting capabilities [99].
• Nuclease Size and Delivery: The physical size of the nuclease is critical for in vivo delivery, especially when using viral vectors like Adeno-Associated Viruses (AAVs). Smaller nucleases like SaCas9 (1053 aa) and AsCas12f1 are favorable for such applications [99] [104].
• Editing Fidelity and Off-Target Effects: Off-target editing is a major concern. High-fidelity (HiFi) variants like eSpOT-ON (an engineered PsCas9) and hfCas12Max are designed to minimize off-target effects while retaining robust on-target activity, which is crucial for both basic research and clinical applications [99] [101].
• Multiplexing Capability: Cas12a possesses inherent ribonuclease activity, allowing it to process a single CRISPR RNA (crRNA) transcript into multiple individual guide RNAs. This simplifies the design of constructs for multiplex genome editing, enabling simultaneous targeting of several genes [103] [102].

Diagram 1: A decision tree for selecting an appropriate CRISPR nuclease based on experimental goals.

Application Notes for Plant Genome Editing

Overcoming the Tissue Culture Bottleneck

A primary challenge in plant genome editing is regenerating whole, edited plants without the lengthy and recalcitrant tissue culture process. Recent breakthroughs are addressing this bottleneck:

• Synthetic Regeneration Systems: A novel approach from Texas Tech University engineers plants to express two key genes—WIND1 (a wound-response trigger) and IPT (involved in hormone production)—directly in wounded tissues. This activates the plant's innate regeneration pathways, allowing edited shoots to grow directly from the plant, bypassing traditional tissue culture [22]. This system has shown success in tobacco, tomatoes, and soybeans.
• In Planta Transformation Methods: Ongoing research focuses on delivering CRISPR components directly to meristematic cells or reproductive organs, aiming to generate edited seeds without any tissue culture step [2]. The synergy between these delivery methods and compact, highly efficient nucleases like ttLbCas12a Ultra V2 or SaCas9 is a key area for future development.

Diagram 2: A simplified workflow for tissue culture-free gene editing in plants using a synthetic regeneration system.

Optimized Experimental Protocols

This protocol is designed for generating stable transgenic plants via Agrobacterium tumefaciens-mediated T-DNA delivery.

• Guide RNA Design and Vector Assembly:

  • For SpCas9: Design 20-nucleotide protospacers preceding a 5'-NGG-3' PAM. For multiplexing, express multiple sgRNAs using individual U6 or U3 promoters.
  • For LbCas12a: Design 20-nucleotide protospacers preceding a 5'-TTTV-3' PAM. For multiplexing, a single array of crRNAs separated by a direct repeat can be used, leveraging Cas12a's own RNA processing ability. A tRNA-based array is also highly effective.
  • Use a GoldenGate modular cloning toolkit to assemble the final expression construct(s).

• Nuclease Expression Cassette:

  • For SpCas9, use a Zea mays codon-optimized sequence with 13 introns (ZmCas9 + 13int) driven by a strong constitutive promoter (e.g., ZmUbi).
  • For LbCas12a, use an Arabidopsis codon-optimized sequence with multiple introns and mutations (e.g., D156R for improved activity). Employ a tandem terminator and ensure the nuclease is flanked by nuclear localization signals (NLS) at both ends.

• Plant Transformation and Selection:

  • Transform barley cultivar 'Golden Promise' or wheat using established Agrobacterium methods.
  • For wheat, co-express GRF-GIF transcription factors to boost transformation efficiency.
  • Select transformed plants (T0) on appropriate antibiotics.

• Molecular Analysis of T0 Plants:

  • Isolate genomic DNA from leaf tissue.
  • Amplify target regions by PCR and subject amplicons to Sanger sequencing.
  • Analyze sequencing chromatograms for indels using tools like TIDE or CRISPResso2. Efficiency is calculated as the percentage of independent T0 lines with mutations at the target locus.

This protocol uses an ultra-optimized LbCas12a variant for high efficiency in dicot plants.

• Plant Material and Growth: Grow Arabidopsis thaliana (e.g., Col-0) under standard conditions (22°C, long-day photoperiod).
• Vector Construction:
  • Use the ttLbCas12a Ultra V2 nuclease variant, which contains the E795L mutation for enhanced catalytic activity and is optimized for lower temperature operation.
  • Clone crRNAs targeting genes of interest (e.g., GL1, CHLI1/CHLI2) into an appropriate expression vector.
• Plant Transformation: Transform plants using the floral dip method.
• Screening and Genotyping:
  • Harvest T1 seeds and select on appropriate antibiotics.
  • Screen resistant plants for edits by PCR and sequencing of the target locus. Homozygous or biallelic mutants can often be identified in the T1 generation with high efficiency.

The Scientist's Toolkit: Essential Reagents

Table 2: Key Research Reagent Solutions for Plant CRISPR Experiments

Reagent / Solution	Function	Example & Notes
Optimized Cas Nuclease	Catalyzes DNA cleavage at target sites.	ZmCas9+13int for cereals; ttLbCas12a Ultra V2 for high-efficiency editing in dicots [102] [103].
Guide RNA Expression System	Directs nuclease to specific genomic locus.	Polymerase III promoters (U6, U3) for single guides; tRNA-based arrays for Cas12a multiplexing [102].
Modular Cloning System	Facilitates rapid vector assembly.	GoldenGate-based toolkits for barley and wheat, available via AddGene [102].
Transformation-Boosting Factors	Increases regeneration efficiency in recalcitrant species.	GRF-GIF co-expression in wheat to maximize transgenic recovery [102].
Synthetic Regeneration System	Enables tissue culture-free editing.	WIND1 + IPT gene combination to induce direct shoot growth from somatic tissue [22].

Discussion and Future Perspectives

The evolution of CRISPR nucleases from the foundational SpCas9 to a diverse toolkit of naturally occurring and engineered variants (SaCas9, Cas12a, hfCas12Max) has dramatically expanded the possibilities for plant genome engineering. The critical factors for success now include not only choosing a nuclease with the appropriate PAM specificity, size, and fidelity but also integrating it with advanced delivery and regeneration strategies.

The future of plant CRISPR research lies in the convergence of several key technologies:

• The development of universal tissue culture-free methods that are robust across a wide range of crop species, leveraging systems like WIND1/IPT [22].
• The continued engineering of next-generation nucleases with minimal off-target effects, expanded PAM recognition, and compact sizes for easier delivery.
• The application of multiplexed editing strategies, for which Cas12a is particularly well-suited, to engineer complex traits by simultaneously modifying multiple genes or pathways [103] [102].

By combining optimized CRISPR systems with transformative regeneration techniques, researchers can accelerate the development of improved crop varieties, thereby addressing pressing challenges in global food security.

The integration of CRISPR-based genome editing with plant tissue culture (PTC) represents a transformative advancement in plant biotechnology. However, the regulatory classification of edited plants often hinges on the presence or absence of foreign DNA (transgenes) in the final product. Transgene-free editing methodologies have emerged as a critical approach to navigate the complex global regulatory landscape, potentially accelerating the commercialization of improved crop varieties. This application note examines current methodologies for generating transgene-free edited plants, their associated protocols, and the evolving policy frameworks governing their use, all within the context of plant tissue culture research.

Transgene-Free Editing Methodologies

The creation of transgene-free edited plants relies on delivering CRISPR components into plant cells without integrating the vector DNA into the plant genome. The following table summarizes the primary delivery methods and their key characteristics.

Table 1: Comparison of Transgene-Free CRISPR Delivery Methods

Method	Mechanism	Key Advantage	Tissue Culture Step	Efficiency/Considerations
Agrobacterium-Mediated Transient Transformation [105] [106]	Uses Agrobacterium to deliver CRISPR DNA temporarily; DNA does not integrate.	Widely applicable to many species; well-established protocol.	Callus/plantlet regeneration from infected explants.	A 2025 method using kanamycin selection reported a 17-fold efficiency increase [105].
Ribonucleoprotein (RNP) Delivery [106] [107]	Direct delivery of pre-assembled Cas9 protein and gRNA complexes.	No foreign DNA involved; reduces off-target effects.	Protoplast isolation and regeneration.	High editing efficiency but plant regeneration from protoplasts remains challenging for many species [107].
Biolistic Delivery (Gene Gun) [106]	Gold particles coated with CRISPR DNA, RNA, or RNPs are shot into cells.	Bypasses the need for Agrobacterium; useful for recalcitrant species.	Regeneration from bombarded embryogenic callus or meristems.	Can cause complex DNA insertions if DNA is used; RNA/RNP delivery is more clean [106].
Viral Vector Delivery [108] [84]	Engineered viruses systemicically deliver CRISPR components.	Can achieve editing without classic tissue culture (in planta).	May bypass or require minimal tissue culture.	Limited cargo capacity; potential bio-safety concerns [84].

Workflow for Transgene-Free Plant Development

The following diagram illustrates the general experimental workflow for developing transgene-free edited plants, integrating tissue culture and molecular analysis steps.

Detailed Experimental Protocol: Agrobacterium-Mediated Transient Expression

This protocol, adapted from Li et al. (2025), details the generation of transgene-free edited citrus plants using kanamycin selection to enhance efficiency [105].

Materials and Reagents

Table 2: Essential Research Reagent Solutions

Reagent/Solution	Function/Description	Key Consideration
CRISPR/Cas9 Construct in a binary vector (e.g., pBIN19)	Provides the gene-editing machinery. The T-DNA contains Cas9 and gRNA expression cassettes.	Use a vector with a plant selection marker (e.g., kanamycin resistance) within the T-DNA for transient selection.
Agrobacterium tumefaciens Strain (e.g., EHA105, GV3101)	Biological vector for delivering the CRISPR construct into plant cells.	The strain must be disarmed and compatible with the binary vector system.
Kanamycin Sulfate	Antibiotic for selecting plant cells that have received the T-DNA.	Used in the culture medium for a short period (3-4 days) to enrich for edited cells [105].
Acetosyringone	Phenolic compound that induces the Agrobacterium vir genes, facilitating T-DNA transfer.	Critical for efficient transformation in co-culture media.
Plant Tissue Culture Media (e.g., MS Basal Medium)	Provides essential nutrients and hormones for plant cell growth and regeneration.	Must be supplemented with appropriate plant growth regulators (auxins, cytokinins) for the target species.

Step-by-Step Procedure

• Vector Preparation: Clone the species-specific gRNA(s) targeting your gene of interest into a binary CRISPR/Cas9 vector.
• Agrobacterium Transformation: Introduce the binary vector into an Agrobacterium strain via freeze-thaw or electroporation.
• Explant Preparation: Aseptically prepare and pre-culture the target explants (e.g., citrus epicotyl segments, leaf discs) on regeneration medium for 2-3 days.
• Agrobacterium Co-culture:
  • Grow the Agrobacterium culture to an OD₆₀₀ of ~0.6-1.0.
  • Pellet the bacteria and resuspend in liquid co-culture medium (e.g., MS salts, sucrose, acetosyringone 100-200 µM) to the final OD₆₀₀.
  • Immerse the explants in the bacterial suspension for 10-30 minutes.
  • Blot dry and transfer the explants to solid co-culture medium. Incubate in the dark at 22-25°C for 2-4 days.
• Transient Selection and Regeneration:
  • After co-culture, transfer explants to regeneration medium containing a suitable concentration of kanamycin (e.g., 100 mg/L) and timentin/carbenicillin (to kill the Agrobacterium).
  • Maintain the explants on this selection medium for a short, critical window of 3-4 days [105]. This eliminates non-transformed cells but allows cells with transient CRISPR expression to survive and be edited.
  • After this period, transfer the explants to the same regeneration medium without kanamycin to allow for shoot regeneration from the edited cells.
• Molecular Screening (T0 Generation):
  • Once regenerated shoots develop, extract genomic DNA.
  • Perform PCR amplification of the target region and sequence the products to identify on-target edits.
  • Use PCR with primers specific to the T-DNA (e.g., Cas9, kanamycin resistance gene) to screen for plants that are free of the transgene.
• Confirmation in Progeny (T1 Generation):
  • Self-pollinate transgene-free, edited T0 plants or cross them with wild-type plants.
  • Analyze the T1 progeny to confirm stable inheritance of the edited trait and the absence of the T-DNA.

Global Regulatory Considerations

The global regulatory landscape for CRISPR-edited plants is fragmented, primarily revolving around whether the product contains foreign DNA. The following diagram outlines the key decision pathways and regulatory outcomes in different jurisdictions.

Table 3: Summary of Global Regulatory Approaches for Transgene-Free Edited Plants

Region	Regulatory Framework	Status of Transgene-Free Edited Plants	Key Implication
United States	USDA SECURE Rule (2020)	Largely exempt from biotechnology regulations if they could have been developed through conventional breeding [107].	Significantly streamlined path to market; treated similarly to conventionally bred crops.
European Union	Proposed Legislation (2024)	To be categorized as NGTs (New Genomic Techniques), with varying levels of regulation based on the modification type [107].	Likely less regulated than transgenic GMOs, but not fully deregulated. Final status pending.
Argentina, Brazil, Japan	Product-Based	Often not considered GMOs if no transgene is present, undergoing a simplified confirmation process [107].	Fosters a more favorable environment for research and development of transgene-free crops.
Other Countries	Mosaic of Regulations	Ranges from permissive to highly restrictive.	Creates challenges for the international trade of edited crops, necessitating case-by-case assessment.

The development of transgene-free CRISPR-edited plants is a pivotal strategy for aligning plant biotechnology innovation with evolving global regulatory policies. The methodologies detailed herein, particularly advanced transient transformation and RNP delivery, provide robust pathways to generate edited crops without persistent transgenes. For researchers in plant tissue culture, mastering these protocols and understanding the associated regulatory frameworks is essential for efficiently translating laboratory breakthroughs into commercially viable, socially acceptable crop varieties that contribute to a sustainable agricultural future.

Benchmarking Against Conventional Breeding and Transgenic Approaches

The advancement of plant breeding technologies has progressively shifted from phenotype-dependent selection to precise genetic manipulation, enabling the direct introduction of desired traits into crops. Within the context of plant tissue culture research, these technologies rely on efficient in vitro systems for plant regeneration and genetic modification. This document provides application notes and detailed protocols for benchmarking conventional breeding, transgenic, and CRISPR-based genome editing approaches. We focus on their applications within plant tissue culture and molecular breeding, emphasizing comparative efficiency, precision, and practical implementation for researchers and scientists engaged in crop improvement and biotechnological development.

Comparative Benchmarking of Breeding Technologies

The following tables provide a quantitative and qualitative comparison of major plant breeding technologies, highlighting their relative performance across multiple criteria.

Table 1: Comparative Analysis of Plant Breeding Technologies

Criterion	Conventional Breeding	Transgenic (GM) Approaches	CRISPR Genome Editing
Precision	Low – relies on phenotype and linkage drag [109]	Moderate – introduces specific genes, but position effects can vary [110]	High – targets specific genes or nucleotides [109]
Speed	Slow – requires multiple generations [109]	Moderate – faster than conventional, but involves complex regulatory processes [111]	Fast – direct edits can reduce breeding cycles from years to months [112] [113]
Trait Specificity	Broad, polygenic traits [109]	Gene-specific, but may have pleiotropic effects [110]	Highly specific, gene-level modifications [109]
Genetic Predictability	Low – segregation may mask traits [109]	Moderate – predictable insertion but variable expression [110]	High – direct and heritable edits [109]
Regulatory Complexity	Low [109]	High – stringent global regulations [112] [113]	Variable – evolving, generally less than GM in some regions [112] [113]
Public Acceptance	High [109]	Low – significant public resistance [110] [114]	Moderate – relatively less public resistance [110] [114]

Table 2: Quantitative Performance Metrics of Breeding Technologies

Parameter	Conventional Breeding	Transgenic Approaches	CRISPR Genome Editing
Typical Development Timeline	7-15 years [115]	5-10 years [115]	2-5 years [112]
Transformation/Editing Efficiency	Not Applicable (N/A)	Varies by species and method (e.g., 15-60% for A. rhizogenes in Liriodendron [116])	High efficiency demonstrated (e.g., 18% in Fraxinus [4], ~100% albinism in banana [111])
Technology Adoption (Market CAGR)	Part of overall market	Part of overall market	9.2% (2025-2030 forecast for biotech methods) [112] [113]
Relative Cost	Low upfront but labor-intensive [109]	High R&D and regulatory costs [112] [113]	High initial investment, cost-effective long-term [109]

Detailed Experimental Protocols

This section provides step-by-step methodologies for key genetic transformation and editing techniques relevant to plant tissue culture systems.

Protocol:Agrobacterium rhizogenes-Mediated Transformation and Gene Editing in Woody Species (e.g., Liriodendron hybrid)

This protocol, adapted from [116], establishes a rapid, efficient hairy root system for functional gene studies in recalcitrant woody plants, bypassing the need for stable plant regeneration.

I. Research Reagent Solutions

Item	Function/Description
Agrobacterium rhizogenes Strains (K599, MSU440, C58C1)	Gram-negative soil bacterium; transfers Root-inducing (Ri) plasmid DNA to plant cells to generate transgenic "hairy roots" [116].
Binary Vector (e.g., pRI101 with eGFP)	Carries gene of interest (GOI) and selectable marker; transferred to plant genome via A. rhizogenes [116].
Murashige and Skoog (MS) Medium	Standard plant tissue culture medium providing essential nutrients for explant growth [116].
Acetosyringone	Phenolic compound added to co-culture medium to induce Vir gene expression in Agrobacterium, enhancing transformation efficiency [116].
Antibiotics (e.g., Kanamycin, Timentin)	Selective agents for controlling bacterial growth post-transformation [116].

II. Step-by-Step Workflow

• Plant Material Preparation:

  • Use two-month-old sterile Liriodendron hybrid seedlings as explants.
  • Make longitudinal incisions at the apical bud to expose cambium cells for infection.

• Agrobacterium Preparation and Inoculation:

  • Transform the binary vector (e.g., containing CRISPR/Cas9 components) into an A. rhizogenes strain (K599 showed highest efficiency ~46% [116]).
  • Grow a single colony in liquid LB medium with appropriate antibiotics at 28°C until OD600 ≈ 0.6.
  • Centrifuge and resuspend the bacterial pellet in liquid MS medium.
  • Apply the bacterial suspension directly to the incised apical bud sites.

• Co-culture and Hairy Root Induction:

  • Co-culture inoculated explants in the dark at 23°C for 2 days.
  • Transfer explants to hormone-free MS solid medium containing antibiotics (e.g., Timentin) to suppress bacterial overgrowth.
  • Hairy roots typically emerge from infection sites within 2-4 weeks.

• Selection and Identification of Transgenic Roots:

  • Select growing roots and screen for fluorescent marker expression (e.g., eGFP).
  • Excise positive roots and subculture on fresh selection medium.

• Molecular Confirmation:

  • Perform PCR and sequencing on genomic DNA from hairy roots to confirm the integration of the T-DNA and the occurrence of targeted gene editing.

III. Application Notes

• This system achieved up to 60.38% transformation efficiency in Liriodendron hybrids [116].
• It enables functional gene validation through overexpression or CRISPR/Cas9-mediated knockout (e.g., targeting the LhAQP1 gene for drought tolerance studies) within months, significantly faster than stable transformation via A. tumefaciens.
• The limitation is that edits are confined to the hairy root tissue and may not be heritable.

Protocol: CRISPR/Cas9-Mediated Genome Editing in Recalcitrant Plants via Meristem Transformation (e.g., Fraxinus mandshurica)

This protocol [4] details a method for achieving heritable gene edits in plant species lacking robust tissue culture and regeneration systems by directly editing meristematic cells.

I. Research Reagent Solutions

Item	Function/Description
Agrobacterium tumefaciens Strain EHA105	Disarmed Agrobacterium strain used for DNA delivery into plant cells via its Tumor-inducing (Ti) plasmid [4].
CRISPR Vector (e.g., pYLCRISPR/Cas9P35S-N)	Binary vector expressing Cas9 nuclease and single guide RNA (sgRNA) under plant-specific promoters [4].
Woody Plant Medium (WPM)	Tissue culture medium optimized for growth of woody plant species [4].
Kanamycin	Antibiotic used as a selective agent for transformed plant tissues [4].
Benzyl Adenine (BA) and Thidiazuron (TDZ)	Plant growth regulators (cytokinins) used to induce and proliferate clustered buds from meristems [4].

II. Step-by-Step Workflow

• Target Selection and Vector Construction:

  • Design sgRNAs targeting the gene of interest (e.g., FmbHLH1).
  • Clone sgRNA sequences into the CRISPR/Cas9 binary vector.
  • Transform the final construct into A. tumefaciens strain EHA105.

• Plant Material and Inoculation:

  • Surface-sterilize Fraxinus mandshurica seeds and germinate on WPM medium for 7 days to obtain sterile plantlets with growing points (meristems).
  • Culture Agrobacterium to OD600 = 0.7, centrifuge, and resuspend in infection medium.
  • Infect the meristematic growing points of sterile plantlets with the Agrobacterium suspension.

• Selection and Induction of Clustered Buds:

  • Co-culture infected plantlets for 3 days.
  • Transfer to selective WPM medium containing kanamycin ( lethal concentration determined to be 50 mg/L) and cytokinins (2.0 mg/L BA + 0.5 mg/L TDZ) to induce the formation of clustered buds from edited meristem cells.

• Screening for Homozygous Edited Plants:

  • After 4-6 weeks, isolate individual shoots from clustered buds.
  • Extract genomic DNA and perform PCR/sequencing to identify lines with homozygous mutations.
  • Regenerate whole plants from edited shoots.

III. Application Notes

• This system achieved an 18% gene editing efficiency in Fraxinus mandshurica meristems [4].
• The clustered bud system enables rapid screening and recovery of homozygous edited plants without going through a lengthy life cycle, crucial for perennial trees.
• This approach is broadly applicable to other plant species with long life cycles or recalcitrant regeneration.

Protocol: Enhanced Biolistic Delivery for CRISPR-Cas RNP in planta Editing

This protocol leverages a novel Flow Guiding Barrel (FGB) device to significantly improve the efficiency and consistency of biolistic delivery, particularly for CRISPR ribonucleoproteins (RNPs) [117].

I. Research Reagent Solutions

Item	Function/Description
Flow Guiding Barrel (FGB)	A 3D-printed device that replaces internal spacer rings in a standard gene gun; optimizes gas and particle flow to increase target area and particle velocity [117].
Gold Microcarriers (0.6 µm)	Tiny, biologically inert particles coated with DNA, RNA, or proteins for ballistic delivery into plant cells [117].
CRISPR-Cas9 Ribonucleoprotein (RNP)	Pre-assembled complex of Cas9 protein and guide RNA; enables DNA-free editing, reducing off-target effects and simplifying regulatory approval [117].
Spermidine	Polyamine used in the precipitation of nucleic acids onto microcarriers [117].

II. Step-by-Step Workflow

• FGB Device Setup:

  • Install the custom FGB into the bombardment chamber of a Bio-Rad PDS-1000/He system, replacing the standard internal barrel and spacer rings.

• Cargo Preparation and Coating:

  • For RNP delivery, pre-assemble the Cas9 protein and sgRNA to form the RNP complex.
  • Precipitate the RNP complexes (or DNA/RNA) onto gold microcarriers using spermidine and calcium chloride.

• Bombardment Parameters:

  • Load the coated microcarriers onto macrocarriers.
  • Use a longer target distance (e.g., 9 cm) and reduced helium pressure (e.g., 650 psi) as optimized for the FGB.
  • Perform bombardment. The FGB generates a more uniform laminar flow, directing nearly 100% of particles to the target with higher velocity and a 4x larger target area.

• Post-Bombardment Culture and Screening:

  • Culture the bombarded tissues (e.g., onion epidermis, maize immature embryos, wheat meristems) under standard conditions.
  • Screen for transient expression or regenerate stable plants and analyze editing events via sequencing.

III. Application Notes

• The FGB increased transient transfection efficiency 22-fold in onion epidermis and CRISPR-Cas9 RNP editing efficiency by 4.5-fold compared to the conventional system [117].
• In maize B104 embryos, stable transformation frequency improved over 10-fold [117].
• This method is ideal for delivering various cargoes (DNA, RNA, RNP, proteins) and is particularly valuable for species or tissues recalcitrant to Agrobacterium infection.

Visualization of Workflows and Relationships

The following diagrams illustrate the logical workflows and technology relationships described in this document.

Conclusion

The convergence of plant tissue culture and CRISPR technology has created a powerful, synergistic platform for precise genetic manipulation in plants. This partnership is fundamental for translating CRISPR breakthroughs from the molecular level into stable, regenerated plants with enhanced traits. Future progress hinges on overcoming persistent challenges in delivery and regeneration efficiency, particularly for recalcitrant species. The emergence of transgene-free editing methods, advanced tools like base editors and CRISPR activation, and evolving regulatory frameworks are poised to accelerate the development of next-generation crops. For biomedical and clinical research, this progress not only promises a more sustainable and secure supply of plant-derived pharmaceuticals but also establishes robust platforms for molecular pharming, where plants can be engineered to produce complex therapeutic compounds, antibodies, and vaccines, thereby expanding the role of plant biotechnology in human health.

References

• 1. Development of a highly efficient protoplast regeneration ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1604283/full]
• 2. Eliminating tissue culture from plant gene editing in the ... [https://www.sciencedirect.com/science/article/pii/S2214662825000015]
• 3. The Technological Trifecta: How Automation, AI, and ... [https://plantcelltechnology.com/blogs/blog/the-technological-trifecta-how-automation-ai-and-crispr-are-forging-the-future-of-plant-science?srsltid=AfmBOoqR3cBO4Jyc6K2hPwjQnN1uJmOvsKNrKf3NtuIaf6mreB0NvpPV]
• 4. Establishment of a CRISPR/Cas9 gene editing system ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12379479/]
• 5. How To Use The Plant Tissue Culture Technology [https://plantcelltechnology.com/blogs/blog/how-to-use-the-tissue-culture-technology?srsltid=AfmBOoqdo4S1Y79YB54xK7Nt7-czK62ybUs-p1TPrEnOadJkVPxWcX53]
• 6. Application of a multiplex CRISPR/Cas9 strategy for ... [https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2025.1633104/full]
• 7. CARBON Newsletter (27 May 2025) - Your Latest ... [https://crisprmedicinenews.com/news/carbon-newsletter-27-may-2025-your-latest-news-about-crispr-in-agrobio/]
• 8. CRISPR-Cas9 Protocol for Efficient Gene Knockout and ... [https://en.bio-protocol.org/en/bpdetail?id=5012&type=0]
• 9. CRISPR-Cas9 Protocol for Efficient Gene Knockout and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11166535/]
• 10. Qualitative and Quantitative Detection of CRISPR ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10572612/]
• 11. Comparing Gene Editing Platforms: CRISPR vs. Traditional ... [https://blog.crownbio.com/comparing-gene-editing-platforms-crispr-vs-traditional-methods]
• 12. Precision gene editing: The power of CRISPR-Cas in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12590234/]
• 13. The Technological Trifecta: How Automation, AI, and ... [https://plantcelltechnology.com/blogs/blog/the-technological-trifecta-how-automation-ai-and-crispr-are-forging-the-future-of-plant-science?srsltid=AfmBOoq0MYo-VMU-rXp9JjzmlpgeAv_gvfzSC-rMD8h68HIc5Tb32v2r]
• 14. A Comparative Review of Genetic Modification Tools for ... [https://nhsjs.com/2025/a-comparative-review-of-genetic-modification-tools-for-crop-improvement-efficiency-scalability-and-usability/]
• 15. Gene Editing Techniques: ZFNs, TALENs or CRISPR? [https://www.news-medical.net/life-sciences/Gene-Editing-Techniques-ZFNs-TALENs-or-CRISPR.aspx]
• 16. Pros and cons of ZNFs, TALENs, and CRISPR/Cas [https://www.jax.org/news-and-insights/jax-blog/2014/march/pros-and-cons-of-znfs-talens-and-crispr-cas]
• 17. CRISPR vs. TALEN vs. ZFN: Which Gene Editing Tool ... [https://synapse.patsnap.com/article/crispr-vs-talen-vs-zfn-which-gene-editing-tool-should-you-use]
• 18. Post-genomic era of CRISPR/Cas technology in ... [https://www.maxapress.com/article/doi/10.48130/opr-0025-0007]
• 19. insights into genome editing and its regulatory overview [https://pubmed.ncbi.nlm.nih.gov/40632351/]
• 20. Nodal culture for efficient regeneration and CRISPR/Cas ... [https://link.springer.com/article/10.1007/s44281-025-00080-9]
• 21. Protoplast-Based Regeneration Enables CRISPR/Cas9 ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12251794/]
• 22. Texas Tech Scientists Develop Novel Acceleration ... [https://www.ttu.edu/now/posts/2025/11/texas-tech-scientists-develop-novel-acceleration-technique-for-crop-creation.php]
• 23. CRISPR/Cas9 editing of CBP80 enhances drought ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1598947/full]
• 24. AI-Driven Advances in Plant Biotechnology [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1718810/full]
• 25. Plant Biotechnology Market Size | Industry Report 2025-2030 [https://www.marketsandmarkets.com/Market-Reports/plant-biotechnology-market-225105561.html]
• 26. Plant Biotechnology Industry Research 2025-2030 [https://finance.yahoo.com/news/plant-biotechnology-industry-research-2025-132500638.html]
• 27. Plant Biotechnology Market Demand and Growth Insights ... [https://www.usdanalytics.com/industry-reports/plant-biotechnology-market]
• 28. CRISPR-Cas9 Application in Crop Biotechnology [https://nhsjs.com/2025/crispr-cas9-application-in-crop-biotechnology-a-decade-of-research-2014-to-2024-toward-food-security-in-developing-countries/]
• 29. Agricultural Biotechnology Market to Promote USD 294.63 ... [https://www.towardshealthcare.com/insights/agricultural-biotechnology-market-sizing]
• 30. Global Gene Editing Market 2025 Growth Top Leaders [https://www.intellectualmarketinsights.com/blogs/global-gene-editing-market-2025-growth-top-leaders]
• 31. Guidelines for Performing CRISPR/Cas9 Genome Editing ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10610170/]
• 32. Strategies for delivery of CRISPR/Cas-mediated genome ... [https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2023.1209586/full]
• 33. Perspectives on the use of the CRISPR system in plants ... [https://www.sciencedirect.com/science/article/abs/pii/S0168165625001348]
• 34. CRISPR/Cas-mediated plant genome editing [https://www.sciencedirect.com/science/article/pii/S1360138523001644]
• 35. Agrobacterium-Mediated Genome Modification for ... [https://www.lidsen.com/journals/genetics/genetics-09-02-292]
• 36. CRISPR/Cas9 in plant biotechnology: applications and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9642946/]
• 37. IMAGE: INTEGRATE-Mediated Agrobacterium Genome ... [https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2025.1676008/full]
• 38. Resources for the design of CRISPR gene editing experiments [https://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0823-x]
• 39. qEva-CRISPR: a method for quantitative evaluation of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6158505/]
• 40. Engineered CRISPR-Cas9 Vector for Efficient ... [https://pubmed.ncbi.nlm.nih.gov/41162805/?utm_source=FeedFetcher&utm_medium=rss&utm_campaign=None&utm_content=1xSjLNP-2lGFmiI8iZJcBJ7eQzwrH7r3HJMuwSCBD7uts8h9bB&fc=None&ff=20251030221730&v=2.18.0.post22+67771e2]
• 41. Protocol for Agrobacterium-mediated transformation of ... [https://www.sciencedirect.com/science/article/pii/S266616672500365X]
• 42. How To Use ICE: A Detailed Guide for Analyzing CRISPR ... [https://www.synthego.com/guide/how-to-use-crispr/ice-analysis-guide/]
• 43. Optimized protocols for protoplast isolation, transfection, and... [https://link.springer.com/article/10.1186/s13765-024-00870-1]
• 44. CRISPR RNP-Mediated Transgene-Free Genome Editing ... [https://pubmed.ncbi.nlm.nih.gov/40923633/]
• 45. A novel protocol for protoplast isolation, transfection, and ... [https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-025-07686-1]
• 46. PEG-mediated transformation and CRISPR/Cas9 gene ... [https://www.sciencedirect.com/science/article/pii/S0926669025012208]
• 47. Generation of transgene - free genome- edited carrot plants using... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12041022/]
• 48. A fast-track system for transgene-free genome editing in oil ... [https://pubmed.ncbi.nlm.nih.gov/41113922/]
• 49. A long shelf-life melon created via CRISPR/Cas9 RNP- ... [https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2025.1623097/full]
• 50. Heritable transgene-free genome editing in plants by ... [https://www.nature.com/articles/s41587-022-01585-8]
• 51. Hairy CRISPR: Genome Editing in Plants Using Hairy Root ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8747350/]
• 52. Highly efficient hairy root genetic transformation and ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1039094/full]
• 53. An efficient hairy root system for validation of CRISPR/Cas ... [https://pubmed.ncbi.nlm.nih.gov/40455261/]
• 54. A simple widely applicable hairy root transformation ... [https://www.sciencedirect.com/science/article/pii/S2211383525001789]
• 55. CRISPR/Cas9 gene editing systems for enhancing ... [https://link.springer.com/article/10.1007/s44372-025-00334-w]
• 56. A High-Throughput, Cost-Effective in Planta Hairy Root ... [https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-025-00990-6]
• 57. Harnessing Agrobacterium rhizogenes and mobile ... [https://www.maxapress.com/article/id/68648267fa6c5854b3ea51cc]
• 58. Injection-based hairy root induction and plant regeneration ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01150-1]
• 59. CRISPR activation: identifying and using novel genes for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12450905/]
• 60. Reference genome and population ... [https://www.nature.com/articles/s41467-025-64825-w]
• 61. iTRAQ-based quantitative proteomic analysis of herbicide ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11696301/]
• 62. CRISPR/Cas9-mediated editing of uORFs in the BnVTC2 ... [https://www.sciencedirect.com/science/article/pii/S2667064X25002726]
• 63. Development and activity evaluation of a highly efficient ... [https://www.sciencedirect.com/science/article/pii/S2468014125001190]
• 64. The Technological Trifecta: How Automation, AI, and ... [https://plantcelltechnology.com/blogs/blog/the-technological-trifecta-how-automation-ai-and-crispr-are-forging-the-future-of-plant-science?srsltid=AfmBOoqSo8SeCNONW830LBx_WyjKFbUQU3P4AQ6Ai1gYtN4GOcQDmLzz]
• 65. Optimizing Agrobacterium-Mediated Transformation and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8535416/]
• 66. CRISPR/Cas-Mediated Optimization of Soybean Shoot ... [https://www.mdpi.com/1422-0067/26/16/7925]
• 67. Review Article CRISPR/Cas genome editing in soybean [https://www.sciencedirect.com/science/article/pii/S2090123224003679]
• 68. Construction of multi-targeted CRISPR libraries in tomato ... [https://www.nature.com/articles/s41467-025-59280-6]
• 69. Optimized protocols for protoplast isolation, transfection, and ... [https://applbiolchem.springeropen.com/articles/10.1186/s13765-024-00870-1]
• 70. Key issues in editing elite-tomato lines by CRISPR-Cas9 ... [https://link.springer.com/article/10.1007/s10681-025-03648-8]
• 71. Comprehensive benchmarking of genome editing ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12144422/]
• 72. Development of a highly efficient protoplast regeneration ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12491312/]
• 73. Genotype-dependent enhancement of tomato ... [https://link.springer.com/article/10.1007/s10725-025-01381-9]
• 74. Unlocking plant regeneration capacity: small signaling ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12554668/]
• 75. Rapid assessment of CRISPR gRNAs with optimized ... [https://pubmed.ncbi.nlm.nih.gov/40938388/]
• 76. Design of highly functional genome editors by modelling ... [https://www.nature.com/articles/s41586-025-09298-z]
• 77. Protocol to rapidly screen CRISPR-Cas9 gene editing ... [https://www.sciencedirect.com/science/article/pii/S2666166725003569]
• 78. A quick-to-implement and optimized CRISPR-Cas9 ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12240085/]
• 79. harnessing multiplex editing for polygenic trait engineering ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12527382/]
• 80. Applications of multiplexed CRISPR–Cas for genome ... [https://www.nature.com/articles/s12276-025-01500-6]
• 81. Programmable Multiplex Genome Editing: Innovations in ... [https://www.sciepublish.com/article/pii/680]
• 82. Complex Trait Loci in Maize Enabled by CRISPR-Cas9 ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.00535/full]
• 83. Crop genome editing through tissue-culture-independent ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11655202/]
• 84. Recent advances of CRISPR-based genome editing for ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1478398/full]
• 85. Off-target effects in CRISPR-Cas genome editing for ... [https://www.sciencedirect.com/science/article/pii/S2162253125001908]
• 86. Revolutionizing CRISPR technology with artificial intelligence [https://www.nature.com/articles/s12276-025-01462-9]
• 87. Highly efficient CRISPR-mediated genome editing through ... [https://www.nature.com/articles/s41467-024-52493-1]
• 88. Optimization of a CRISPR-Cas9 in vitro protocol for ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1523884/full]
• 89. Optimize Your CRISPR Experiments Like a Pro [https://www.synthego.com/blog/crispr-optimization/]
• 90. Advances and Applications of Plant Base Editing Technologies [https://pmc.ncbi.nlm.nih.gov/articles/PMC12524351/]
• 91. New strategies to advance plant transformation [https://www.sciencedirect.com/science/article/pii/S0958166924001770]
• 92. Validating CRISPR/Cas9-mediated Gene Editing [https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/advanced-gene-editing/validating-crispr-cas9-mediated-gene-editing?srsltid=AfmBOopcMSjqyehnNZEsN3J4aeida9cFloQASBNfNEqeoI9JBCm788lD]
• 93. Verification of CRISPR Gene Editing Efficiency [https://www.thermofisher.com/us/en/home/life-science/genome-editing/genome-editing-learning-center/genome-editing-resource-library/crispr-validated-protocols/verification-crispr-gene-editing-efficiency.html]
• 94. Detection method for CRISPR-Cas9 edited tomato with ... [https://www.sciencedirect.com/science/article/pii/S1687157X25001532]
• 95. Streamlined protoplast transfection system for in-vivo ... [https://pubmed.ncbi.nlm.nih.gov/40459690/]
• 96. CRISPR activation: identifying and using novel genes for ... [https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2025.1596600/full]
• 97. CRISPR screens in plants: approaches, guidelines, and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8226290/]
• 98. Tiny CRISPR Tool Opens the Door to Faster, Simpler Plant ... [https://innovativegenomics.org/news/tiny-crispr-tool-plant-genome-editing/]
• 99. How to Choose the Right Cas Variant for Every CRISPR ... [https://www.synthego.com/guide/how-to-use-crispr/cas-nuclease-variants/]
• 100. Novel CRISPR–Cas Systems: An Updated Review of the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8036902/]
• 101. Assessing and advancing the safety of CRISPR-Cas tools [https://www.nature.com/articles/s41467-023-35886-6]
• 102. An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01234-y]
• 103. Optimization of CRISPR/Cas12 tools for plant genome editing [https://pmc.ncbi.nlm.nih.gov/articles/PMC12418774/]
• 104. Comparison of CRISPR-Cas9, CRISPR-Cas12f1, and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12131857/]
• 105. New Method Makes Transgene-Free Gene Editing Even More ... [https://today.uconn.edu/2025/11/new-method-makes-transgene-free-gene-editing-even-more-promising/]
• 106. CRISPR in Agriculture [https://innovativegenomics.org/crisprpedia/crispr-in-agriculture/]
• 107. Frontiers in global regulatory landscape of CRISPR-edited ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10851166/]
• 108. The Technological Trifecta: How Automation, AI, and ... [https://plantcelltechnology.com/blogs/blog/the-technological-trifecta-how-automation-ai-and-crispr-are-forging-the-future-of-plant-science?srsltid=AfmBOoq1cASOuNrJygwqdEVRQ1LsGDTdEYH7nyKGiWWdPQxS_V9jWkNN]
• 109. The dilemma of food genetics and improvement - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC12326302/]
• 110. Genetic modification techniques in plant breeding [https://www.sciencedirect.com/science/article/pii/S2468014124001675]
• 111. Efficient CRISPR/Cas9-mediated genome editing of ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1677409/full]
• 112. Plant Breeding and CRISPR Plants Market [https://www.marketsandmarkets.com/Market-Reports/plant-breeding-crispr-plants-market-256910775.html]
• 113. Plant Breeding and CRISPR Plants Market by Type ... [https://www.researchandmarkets.com/reports/6062613/plant-breeding-crispr-plants-market-type?srsltid=AfmBOop3yzB8jQ41briS3nuI2RrLZWJ38_yoGIo0sujoIlEKrDqKHUW8]
• 114. A comparative review of CRISPR/Cas and GM technologies [https://ui.adsabs.harvard.edu/abs/2025HorPJ..11.1807H/abstract]
• 115. Potential of conventional and new breeding technologies to ... [https://phytopatholres.biomedcentral.com/articles/10.1186/s42483-025-00338-0]
• 116. An efficient genetic transformation and gene editing system ... [https://www.sciencedirect.com/science/article/abs/pii/S0176161725002214]
• 117. Enhancing biolistic plant transformation and genome ... [https://www.nature.com/articles/s41467-025-60761-x]